Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Received: 22 May 1996 / Accepted: 24 November 1996     

Mouse beta thalassemia, a model for the membrane defects of erythrocytes in the human disease

The present study describes the pathophysiology, at the cellular level, of the mouse beta thalassemia and shows the pertinence of this model for the human disease. The homozygous state of mouse beta thalassemia is characterized by a clinical syndrome similar to the human beta thalassemia intermedia, but it cannot be explained by the small deficiency in beta chain synthesis. The small pool of unpaired and soluble alpha chains present in mouse reticulocytes contrasts with the large amount of insoluble alpha chains in erythrocytes which is induced by the high instability of mouse alpha chains and the absence of significant proteolysis. The amount of insoluble alpha chains associated with red cell ghosts is similar in human and mouse disease of similar severity. The study of membrane protein defects showed a decreased amount of spectrin (alpha and beta chains) and dramatic changes in the distribution of the most reactive thiol groups of membrane proteins. These results were similar to that previously described in the human disease (Rouyer-Fessard, P., Garel, M. C., Domenget, C., Guetarni, D., Bachir, D., Colonna, P., and Beuzard, Y. (1989) J. Biol. Chem. 264, 19092-19098). Abnormal density distribution curves of erythrocytes and oxidant-induced lysis of red blood cells used as functional tests were similar in the human and mouse beta thalessemia. We conclude from the present study that 1) mouse beta thalassemia is an excellent model for the membrane defects occurring in the human disease; 2) disease expression is not the reflection of the globin chain unbalance only nor of the soluble pool of alpha hemoglobin chain but mainly is a reflection of insoluble alpha chains; and 3) the rate of proteolysis and instability of alpha chains are important factors which must be taken into consideration in the pathophysiology and the clinical heterogeneity of the disease.     

Structural analysis of the gene for the beta chain of the antigen-specific receptor of mammalian T-lymphocytes

The antigen specific receptor of T cells (TCR) is composed of alpha and beta chains and is normally present on the T cell surface complexed with the components which make up T3. In the case of beta chain, multiple somatic DNA rearrangements bring together V beta (variable), D beta (diversity) and J beta (joining) gene segments before a mature messenger RNA can be transcribed. So far beta chain genes have been extensively studied in the human and in the mouse system and we have very little information on other mammals. Our aims were to obtain information that may provide a structural basis for understanding developmental as well as evolutionary aspects of the TCR gene system in mammals. In this study we compare the hybridization pattern between a human cDNA probe coding for the beta chain constant region and restricted genomic DNA extracted from lymphocytes deriving from human as well as from rat and lamb. The comparison of the hybridization data represent a first piece of information about the variation of the structure of the TCR beta chain genes in mammals.     

Intron Distribution in Ancient Paralogs Supports Random Insertion and Not Random Loss

The intron positions of ten different protein families were examined to determine (the statistical likelihood of) whether spliceosomal introns are the result of random insertion events into previously intronless genes, on the one hand, or the result of random loss from common ancestral introns, on the other. The number of expected matches for the alternative scenarios was calculated for a binomial distribution by considering currently observed introns relative to all possible locations for insertion or loss. Introns occurring at approximately the same location (hereafter called a ``match') were tallied for each of the paired proteins. Matches were identified by their positions in the multiple alignment and were defined as any two introns occurring within a window of 11 possible nucleotide positions, thereby allowing for possible alignment errors and ``intron sliding.' Matches were tallied from the raw data and compared with the expected number of matches for the two different scenarios. The results suggest that the distribution of introns in genes encoding proteins is due to random insertion and not random loss. Received: 8 September 1996 / Accepted: 24 January 1997     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains. Received: 5 January 1996 / Accepted: 7 March 1996     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996     

In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.     

A theoretical method for evaluating the relative importance of positive selection and neutral drift from observed base changes

To evaluate the relative importance of positive selection and neutral drift from the nucleotide base changes observed in the homologous alignment of genes, a theoretical equation of base changes is formulated by including both the influence of selection and the base substitutions due to mutations. Under the assumption that the average rate of base substitutions estimated from synonymous changes is the ``true' mutation rate applicable at all positions, this method is applied to the vertebrate globin gene family, and evaluates the departures of base change rates from the ``true' mutation rate at the first and second codon positions as a consequence of preferential selection for the conservation of important function. In addition to the strong effect of selection on the amino acid residues in the internal region mostly common to myoglobin and hemoglobin chains, the distinctive directions of selective parameter values are seen at sites on the globin surface, distinguishing the subunit contact residues of hemoglobins from the polar residues on the surface of myoglobins. Moreover, this effect of selection distinguishing between the myoglobin and hemoglobin chain genes becomes weaker in cold-blooded vertebrates, especially in fish, strongly suggesting the possibility that the clear distinction between these globins is a result of selection out of the changes regarded as neutral ones in an ancestor of vertebrates. Thus, the present method may also serve to investigate the homology of many other proteins from the aspect of molecular evolution, mainly focusing on the evolution of their biological functions. Received: 2 January 1996 / Accepted: 20 February 1997     

Current Intraspecific Dynamics of Sequence Evolution Differs from Long-Term Trends and Can Account for the AT-Richness of Honeybee Mitochondrial DNA

The very high AT content of hymenopteran mtDNA has warranted speculation about nucleotide substitution processes in this group. Here we investigate the pattern of honeybee, Apis mellifera, mtDNA nucleotide polymorphisms inferred from phylogeny in terms of differences between the ATPase6, COI, COII, COIII, cytochrome b, and ND2 genes and strand asymmetry in mutation rates. The observed transition/transversion ratios and the distribution of nonsynonymous substitutions between regions differed significantly. The pattern of differences between genes leading to these heterogeneities (the ATPase6 and COIII genes group apart from the rest) differed markedly from that predicted on the basis of long-term evolutionary change and may indicate differences between current and long-term dynamics of sequence evolution. Also, there is strong strand asymmetry in substitutions, which probably results in a mutability of G and C sufficiently high to account for the AT-richness of honeybee mtDNA. Received: 21 October 1998 / Accepted: 27 January 1999     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Cysteine Mapping in the Ion Selectivity and Toxin Binding Region of the Cardiac Na+ Channel Pore

Aqueous exposure of critical residues in the selectivity region of voltage gated Na⁺ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na⁺ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA⁺) and (2-sulfonatoethyl)methanethiosulfonate (MTSES⁻) in all six of the Cys-substituted channels, including Trp → Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10². Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES⁻ suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES⁻ markedly increased, and MTSEA⁺ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp¹⁷¹³ acts electrostatically in an unrestricted region. These results suggest that Lys¹⁴¹⁸ lies in a restricted region favorable to cations, whereas Asp¹⁷¹³ is at a more peripheral location in the Na⁺ channel pore. Received: 8 May 1996/Revised: 15 August 1996     

Random length assortment of human and mouse T cell receptor for antigen alpha and beta chain CDR3.

In view of the recently determined three-dimensional structures of complexes formed by the T cell receptor for antigen (TCR), the processed peptide and the MHC class I molecule, it is expected that the combined configuration formed by the third complementarity determining regions (CDR3) of TCR alpha and beta chains will be very restricted in size and shape due to the limited length variations of the processed peptides. Thus, the combined TCR alpha and beta chain CDR3 lengths should have a fairly narrow distribution. This feature can be due to the selective association of long alpha chain CDR3 with short beta chain CDR3 and vice versa or due to random assortment of alpha and beta chain CDR3 of even narrower length distribution. Based on existing translated amino acid sequence data, it has been found that the latter mechanism is responsible.     

Analysis of Orthologous Retrovirus-Like Elements in the White-Footed Mouse,Peromyscus leucopus

Three loci in the genome of the white-footed mouse, Peromyscus leucopus, were examined for the presence or absence of orthologous copies of the retrovirus-like element mys using polymerase chain reaction. We examined these loci in 28 mice collected throughout the P. leucopus species range. Mys insertions were present in only one of the individuals examined at the mys-1 and mys-7 loci. Conversely, the mys-6 element was found in several individuals, but the presence of this element was limited to northern latitudes. Because the long terminal repeats (LTRs) of a given element are expected to be identical at the time of retrotransposition into the genome, and to accumulate changes over evolutionary time, within-element LTR sequence comparisons can be used to estimate the relative age of insertions. Within-element LTR differences are greater in mys-6 than in mys-1 or mys-7. The LTRs from orthologous mys-6 elements of six mice were sequenced. The alignment revealed 13 of the 22 differences between the right and left LTRs that were shared by all orthologous mys-6 sites, suggesting that relative to its time of insertion into the genome, mys-6 has only recently spread across the northern part of the species range. Received: 23 January 1996 / Accepted: 24 April 1996     

Horizontal transfer of genes coding for the photosynthetic reaction centers of purple bacteria

Phylogenetic trees were drawn and analyzed based on the nucleotide sequences of the 1.5-kb gene fragment coding for the L and M subunits of the photochemical reaction center of various purple photosynthetic bacteria. These trees are mostly consistent with phylogenetic trees based on 16S rRNA and soluble cytochrome c, but differ in some significant details. This inconsistency implies horizontal transfer of the genes that code for the photosynthetic apparatus in purple bacteria. Possibilities of similar transfers of photosynthesis genes during the evolution of photosynthesis are discussed especially for the establishment of oxygenic photosynthesis. Received: 8 July 1996 / Accepted: 12 March 1997     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

T cell differentiation model based on the expression of T cell receptor chains and genes--study in the human leukemia/lymphoma cell lines

A total of 33 human leukemia/lymphoma cell lines were classified into 4 groups with respect to the pattern of cell membrane (sm) expression of the CD3 and T cell receptor (TCR) molecules; (i) smCD3+TCR alpha beta (16 cell lines), (ii) smCD3+TCR beta delta (1 cell line), (iii) smCD3+TCR gamma delta (3 cell lines) amd (iv) smCD3-TCR- (13 cell lines), respectively. Using monoclonal antibodies (MoAbs) specific to CD3 (NU-T3), TCR alpha chain (alpha F1), TCR beta chain (beta F1), and TCR gamma chain (C gamma M1), respectively, cytoplasmic (cy) expression of these molecules was determined by immunofluorescence test. Expression of cyCD3 was present in all cell lines regardless of groups. In group (i), all 16 cell lines expressed both TCR alpha and beta chains. While only TCR beta chain was expressed in group (ii), TCR gamma chain was expressed in all 3 cell lines of group (iii). One (PEER) of the three in group (iii) expressed TCR beta chain as well. In group (iv), we found 8 cell lines with cyTCR alpha expression, 11 cell lines with cyTCR beta expression, and 10 cell lines with cyTCR gamma expression, respectively. For TCR genes, except 1 cell line all cell lines were found to present rearranged C beta gene and its mRNA, including all 3 TCR gamma/delta cell lines of group (iii). One of the TCR alpha beta cell lines exhibited rearranged C delta and J delta genes as well as its mRNA. Two cell lines of the 13 CD3-TCR- of group (iv) exhibited rearranged C delta and J delta and its mRNA. An NK-like activity and IL-2 production were induced in the TCR beta delta and gamma delta cell lines [group (ii) and (iii)] by treatment with PHA and PMA.