Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Mitochondrial DNA polymorphism among five Asian populations

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely related to each other. Aeta was found to show a relatively close relationship to these three populations, confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different from the other four populations.     

The number of nucleotides required to determine the branching order of three species,with special reference to the human-chimpanzee-gorilla divergence

Summary A mathematical theory for computing the probabilities of various nucleotide configurations among related species is developed, and the probability of obtaining the correct tree (topology) from nucleotide sequence data is evaluated using models of evolutionary trees that are close to the tree of mitochondrial DNAs from human, chimpanzee, gorilla, orangutan, and gibbon. Special attention is given to the number of nucleotides required to resolve the branching order among the three most closely related organisms (human, chimpanzee, and gorilla). If the extent of DNA divergence is close to that obtained by Brown et al. for mitochondrial DNA and if sequence data are available only for the three most closely related organisms, the number of nucleotides (m^*) required to obtain the correct tree with a probability of 95% is about 4700. If sequence data for two outgroup species (orangutan and gibbon) are available, m^* becomes about 2600–2700 when the transformed distance, distance-Wagner, maximum parsimony, or compatibility method is used. In the unweighted pair-group method, m^* is not affected by the availability of data from outgroup species. When these five different tree-making methods, as well as Fitch and Margoliash's method, are applied to the mitochondrial DNA data (1834 bp) obtained by Brown et al. and by Hixson and Brown, they all give the same phylogenetic tree, in which human and chimpanzee are most closely related. However, the trees considered here are gene trees, and to obtain the correct species tree, sequence data for several independent loci must be used.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse

The noncoding region between tRNA^Pro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2–4 × 10^–8 per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.Correspondence to: N. Ishida     

Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest M_r were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher M_r bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest M_r band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher M_r bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower M_r bands of occludin (non- or less phosphorylated form) but was specific to the higher M_r bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower M_r bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Similarity-Based Analysis of Allele Frequency Distribution among Multiple Populations Identifies Adaptive Genomic Structural Variants

Structural variants have a considerable impact on human genomic diversity. However, their evolutionary history remains mostly unexplored. Here, we developed a new method to identify potentially adaptive structural variants based on a similarity-based analysis that incorporates genotype frequency data from 26 populations simultaneously. Using this method, we analyzed 57,629 structural variants and identified 576 structural variants that show unusual population differentiation. Of these putatively adaptive structural variants, we further showed that 24 variants are multiallelic and overlap with coding sequences, and 20 variants are significantly associated with GWAS traits. Closer inspection of the haplotypic variation associated with these putatively adaptive and functional structural variants reveals deviations from neutral expectations due to: 1) population differentiation of rapidly evolving multiallelic variants, 2) incomplete sweeps, and 3) recent population-specific negative selection. Overall, our study provides new methodological insights, documents hundreds of putatively adaptive variants, and introduces evolutionary models that may better explain the complex evolution of structural variants.