Reverse polarization in amino acid and nucleotide substitution patterns between human-mouse orthologs of two compositional extrema.

Genome-wide analysis of sequence divergence patterns in 12,024 human-mouse orthologous pairs reveals, for the first time, that the trends in nucleotide and amino acid substitutions in orthologs of high and low GC composition are highly asymmetric and polarized to opposite directions. The entire dataset has been divided into three groups on the basis of the GC content at third codon sites of human genes: high, medium, and low. High-GC orthologs exhibit significant bias in favor of the replacements, Thr --> Ala, Ser --> Ala, Val --> Ala, Lys --> Arg, Asn --> Ser, Ile --> Val etc., from mouse to human, whereas in low-GC orthologs, the reverse trends prevail. In general, in the high-GC group, residues encoded by A/U-rich codons of mouse proteins tend to be replaced by the residues encoded by relatively G/C-rich codons in their human orthologs, whereas the opposite trend is observed among the low-GC orthologous pairs. The medium-GC group shares some trends with high-GC group and some with low-GC group. The only significant trend common in all groups of orthologs, irrespective of their GC bias, is (Asp)(Mouse) --> (Glu)(Human) replacement. At the nucleotide level, high-GC orthologs have undergone a large excess of (A/T)(Mouse) --> (G/C)(Human) substitutions over (G/C)(Mouse) --> (A/T)(Human) at each codon position, whereas for low-GC orthologs, the reverse is true.     

Apparent heterogeneity of hepatic lysosomes due to membrane-bound acid phosphatase.

Hepatic lysosomes have been fractionated by rate sedimentation and by isopycnic banding. In all experiments, the distribution of acid phosphatase differed from that of the other lysosomal enzymes. Evidence is presented that this difference is due not to the separation of lysosomes from different cell types, but simply reflects the membrane location of a part of the acid phosphatase.     

Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.     

Hexokinase isoenzymes: antigenic cross-reactivities and amino acid compositional relatedness

Previously undetected antigenic cross-reactivities have been demonstrated between yeast and rat hexokinase isoenzymes using an enzyme-linked immune sorbent assay. The levels of structural homology between the isoenzymes have been assessed in terms of their relative antigenic cross-reactivities and their amino acid compositional relatedness. The three major rat hexokinases appear, despite their differing molecular sizes, to have a close evolutionary background and to share a common but distant ancestry with yeast and wheat-germ forms.     

The complete mitochondrial sequence of Tarsius bancanus: evidence for an extensive nucleotide compositional plasticity of primate mitochondrial DNA

Inconsistencies between phylogenetic interpretations obtained from independent sources of molecular data occasionally hamper the recovery of the true evolutionary history of certain taxa. One prominent example concerns the primate infraordinal relationships. Phylogenetic analyses based on nuclear DNA sequences traditionally represent Tarsius as a sister group to anthropoids. In contrast, mitochondrial DNA (mtDNA) data only marginally support this affiliation or even exclude Tarsius from primates. Two possible scenarios might cause this conflict: a period of adaptive molecular evolution or a shift in the nucleotide composition of higher primate mtDNAs through directional mutation pressure. To test these options, the entire mt genome of Tarsius bancanus was sequenced and compared with mtDNA of representatives of all major primate groups and mammals. Phylogenetic reconstructions at both the amino acid (AA) and DNA level of the protein-coding genes led to faulty tree topologies depending on the algorithms used for reconstruction. We propose that these artifactual affiliations rather reflect the nucleotide compositional similarity than phylogenetic relatedness and favor the directional mutation pressure hypothesis because: (1) the overall nucleotide composition changes dramatically on the lineage leading to higher primates at both silent and nonsilent sites, and (2) a highly significant correlation exists between codon usage and the nucleotide composition at the third, silent codon position. Comparisons of mt genes with mt pseudogenes that presumably transferred to the nucleus before the directional mutation pressure took place indicate that the ancestral DNA composition is retained in the relatively fossilized mtDNA-like sequences, and that the directed acceleration of the substitution rate in higher primates is restricted to mtDNA.     

Arachnid relationships based on mitochondrial genomes: asymmetric nucleotide and amino acid bias affects phylogenetic analyses

Phylogenetic analyses based on mitochondrial DNA have yielded widely differing relationships among members of the arthropod lineage Arachnida, depending on the nucleotide coding schemes and models of evolution used. We enhanced taxonomic coverage within the Arachnida greatly by sequencing seven new arachnid mitochondrial genomes from five orders. We then used all 13 mitochondrial protein-coding genes from these genomes to evaluate patterns of nucleotide and amino acid biases. Our data show that two of the six orders of arachnids (spiders and scorpions) have experienced shifts in both nucleotide and amino acid usage in all their protein-coding genes, and that these biases mislead phylogeny reconstruction. These biases are most striking for the hydrophobic amino acids isoleucine and valine, which appear to have evolved asymmetrical exchanges in response to shifts in nucleotide composition. To improve phylogenetic accuracy based on amino acid differences, we tested two recoding methods: (1) removing all isoleucine and valine sites and (2) recoding amino acids based on their physiochemical properties. We find that these methods yield phylogenetic trees that are consistent in their support of ancient intraordinal divergences within the major arachnid lineages. Further refinement of amino acid recoding methods may help us better delineate interordinal relationships among these diverse organisms.     

Elimination of amino acid losses with protein acid hydrolyzates due to adsorption

A relatively convenient and rapid chromatographic method has been devised for the preparation of gram quantities of the main component of human hemoglobin. The product of this preparative method has been examined for the presence of minor hemoglobin components, ferrihemoglobin, and phosphates. The amounts of each of these materials were found to be small. Measurements of the oxygen-binding isotherm and tetramer-dimer dissociation constant of the product were also carried out, and its behavior was found to be consistent with that observed by other investigators for unfractionated, phosphate-free hemoglobin.     

The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice.     

Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells

We have examined the state of methylation of mitochondrial ribosomal RNA from cultured hamster (BHK-21) cells. Ethidium-sensitive, and hence mitochondrion-specific, methylation levels were determined using multiple isotope techniques and improved purification procedures. The larger mitochondrial rRNA species, 17 S RNA, was found to contain 0.13 methyl group per 100 nucleotides and the smaller, 13 S RNA, 0.37. Methylated nucleotide and base analysis indicated that 17 S RNA contained one ribose-methylated residue (UmUp) per molecule and one unidentified residue; and 13 S RNA contained one methylated cytosine residue, one N⁶-dimethyladenine residue and one thymine residue per molecule. Possible evolutionary implications of these findings have been discussed.     

Models of amino acid substitution and applications to mitochondrial protein evolution

Models of amino acid substitution were developed and compared using maximum likelihood. Two kinds of models are considered. "Empirical" models do not explicitly consider factors that shape protein evolution, but attempt to summarize the substitution pattern from large quantities of real data. "Mechanistic" models are formulated at the codon level and separate mutational biases at the nucleotide level from selective constraints at the amino acid level. They account for features of sequence evolution, such as transition-transversion bias and base or codon frequency biases, and make use of physicochemical distances between amino acids to specify nonsynonymous substitution rates. A general approach is presented that transforms a Markov model of codon substitution into a model of amino acid replacement. Protein sequences from the entire mitochondrial genomes of 20 mammalian species were analyzed using different models. The mechanistic models were found to fit the data better than empirical models derived from large databases. Both the mutational distance between amino acids (determined by the genetic code and mutational biases such as the transition-transversion bias) and the physicochemical distance are found to have strong effects on amino acid substitution rates. A significant proportion of amino acid substitutions appeared to have involved more than one codon position, indicating that nucleotide substitutions at neighboring sites may be correlated. Rates of amino acid substitution were found to be highly variable among sites.      

Apparent trends of amino Acid gain and loss in protein evolution due to nearly neutral variation

It has recently been claimed that certain amino acids have been increasing in frequency in all living organisms for most of the history of life on earth, while other amino acids have been decreasing in frequency. Three lines of evidence have been offered for this assertion, but each has a more plausible alternative interpretation. Here I show that unequal patterns of gains and losses for particular pairs of amino acids (such as more leucine --> phenylalanine than phenylalanine --> leucine substitutions in humans and chimpanzees since they split from a common ancestor) are consistent with a simple neutral model at equilibrium amino acid frequencies. Unequal numbers of gains and losses for particular amino acids (such as more gains than losses of cysteine) are shown by simulations to be consistent with a model of nearly neutral evolution. Unequal numbers of gains and losses for particular amino acids in human polymorphism data are shown by simulations to be explainable by the nearly neutral model as well. In a comparison of protein sequences from four strains of Escherichia coli, polarized by one outgroup strain of Salmonella, the disparity in number of gains and losses for particular amino acids is strong in terminal branches but weaker or nonexistent in internal branches, which is inconsistent with the universal trend model but as expected under the nearly neutral model.     

Resistance to l-methionine-S-sulfoximine in Chlamydomonas reinhardtii is due to an alteration in a general amino acid transport system

It was suggested that the mutant ARF1 of Chlamydomonas reinhardtii is resistant to l-methionine-S-sulfoximine (MSX, an irreversible inhibitor of glutamine synthetase, EC 6.3.1.2) because this strain degraded and utilized this compound as a nitrogen source for growth (A.R. Franco et al., 1996, Plant Physiol 110: 1215–1222). Resistance to MSX has now been characterized in a double mutant of this alga, called MPA1, which is resistant to MSX and lacks l-amino acid oxidase (LAO activity, EC 1.4.3.2). Biochemical and genetic evidence indicate that the mutant MPA1 is altered in the same MSX-resistance locus as mutant ARF1. However, mutant MPA1 neither degraded nor utilized MSX as a nitrogen source. This led us to conclude that (i) resistance to MSX is not linked to its utilization, and (ii) that LAO activity accounts for the degradation of MSX in mutant ARF1. Data indicate that C. reinhardtii possesses a broad-specificity carrier system responsible for the transport of arginine and other amino acids, including MSX. We propose that the alteration of this carrier confers resistance to MSX in mutants ARF1 and MPA1. Received: 6 April 1998 / Accepted: 8 June 1998     

Molecular mechanisms involved in the adaptation to amino acid limitation in mammals

In mammals, metabolic adaptations are required to cope with episodes of protein deprivation and malnutrition. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of the expression of numerous genes. In particular, it has been shown that amino acids by themselves can modify the expression of target genes. This review describes the regulation of amino acids homeostasis and the their role as signal molecules. The recent advances in the understanding of the molecular mechanisms involved in the control of mammalian gene expression in response to amino acid limitation will be described.     

Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor

The Hsp70 protein switches during its functional cycle from an ADP-bound state with a high affinity for substrates to a low-affinity, ATP-bound state, with concomitant release of the client protein. The rate of the chaperone cycle is regulated by co-chaperones such as nucleotide exchange factors that significantly accelerate the ADP/ATP exchange. Mge1p, a mitochondrial matrix protein with homology to bacterial GrpE, serves as the nucleotide exchange factor of mitochondrial Hsp70. Here, we analyze the influence of temperature on the structure and functional properties of Mge1p from the yeast Saccharomyces cerevisiae. Mge1p is a dimer in solution that undergoes a reversible thermal transition at heat-shock temperatures, i.e. above 37 degrees C, that involves protein unfolding and dimer dissociation. The thermally denatured protein is unable to interact stably with mitochondrial Hsp70, and therefore is unable to regulate its ATPase and chaperone cycle. Crosslinking of wild-type mitochondria reveals that Mge1p undergoes the same dimer to monomer temperature-dependent shift, and that the nucleotide exchange factor does not associate with its Hsp70 partner at stress temperatures (i.e. > or =45 degrees C). Once the stress conditions disappear, Mge1p refolds and recovers both structure and functional properties. Therefore, Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins. The thermosensor activity is conserved in the GrpE-like nucleotide exchange factors although, as discussed here, it is achieved through a different structural mechanism.