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Biomedical subjects

W H Li

Publications and source records attributed to W H Li.

At least 19 recordsLinked to original sources

Adaptive evolution of color vision genes in higher primates.

The intron 4 sequences of the three polymorphic alleles at the X-linked color photo-pigment locus in the squirrel monkey and the marmoset reveal that the alleles in each species are exceptionally divergent. The data further suggest either that each triallelic system has arisen independently in these two New World monkey lineages, or that in each species at least seven deletions and insertions (14 in the two species) in intron 4 have been transferred and homogenized among the alleles by gene conversion or recombination. In either case, the alleles in each species apparently have persisted more than 5 million years and probably have been maintained by overdominant selection.

Alleles

Alternative mRNA splicing and differential promoter utilization determine tissue-specific expression of the apolipoprotein B mRNA-editing protein (Apobec1) gene in mice. Structure and evolution of Apobec1 and related nucleoside/nucleotide deaminases.

Apolipoprotein (apo) B mRNA editing consists of a C-->U conversion involving the first base of the codon CAA, encoding Gln 2153, to UAA, a stop codon. Editing occurs in the intestine only in most mammals, and in both the liver and intestine in a few mammalian species including mouse. We have cloned the cDNA for the mouse apoB mRNA editing protein, apobec1. Expression of mouse apobec1 cDNA in HepG2 cells results in the editing of the intracellular apoB mRNA. The cDNA predicts a 229-amino acid protein showing 92, 66, and 70% identity to the rat, rabbit, and human proteins, respectively. Based on the estimated values of divergence of apobec1 sequences in terms of the numbers of synonymous and non-synonymous suhstitutions per site, we found that apobec1 is a fairly rapidly evolving protein. Sequence comparison among mammalian apobec1 sequences has permitted the identification of seven conserved regions that may be functionally important for editing activity. We present a phylogenetic tree relating apobec1 sequences to double-stranded RNA adenosine deaminase and other nucleotide/nucleoside deaminases. Northern blot analysis indicates that apobec1 mRNA exists in two different sizes, a approximately 2.2-kilobase (kb) form in small intestine and a approximately 2.4-kb form in liver, spleen, kidney, lung, muscle, and heart. To study the molecular basis for the different sized apobec1 mRNAs, we cloned the apobec1 gene and characterized its exon-intron organization together with the sequences expressed in the hepatic and intestinal mRNA. The mouse apobec1 gene contains 8 exons and spans approximately 25 kb, and is located in chromosome 6. The major hepatic mRNA contains all 8 exons, whereas the major small intestinal mRNA misses the first 3 exons and its transcription is initiated in exon 4. The intestinal mRNA also contains at its 5' end a unique 102-nucleotide piece that is absent in the liver mRNA. We also identified two alternatively spliced hepatic apobec1 mRNAs with different acceptor sites in exon 4. Transient expression studies using promoter-reporter gene constructs in HeLa, Hepa, and Caco-2 cells indicate that the 5'-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity and the 5'-flanking sequences of the major small intestine mRNA (i.e. upstream of exon 4) have preferential intestinal promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

APOBEC-1 Deaminase

Estimation of confidence in phylogeny: the complete-and-partial bootstrap technique.

The bootstrap is a statistical technique that is widely used to assess confidence limits on phylogenies. We show that the power of the bootstrap test is lower than those of the C and S tests suggested by Felsenstein, unless the critical value employed in the bootstrap test is correctly selected. If the 95% critical value is used, the bootstrap proportions are underestimates of the confidence level when the number of possible alternative topologies is three or more; the degree of underestimation increases with the number of competing alternative topologies. To overcome this problem, we propose the complete-and-partial bootstrap technique as a method for obtaining an unbiased estimate of the confidence level. The method is based on a multinomial model of many alternatives among which the choice is to be made. The complete-and-partial bootstrap technique can be used to estimate the effective number of competing alternative topologies and the confidence level of the monophyly of a particular group of taxa or of an inferred tree topology. This approach can be used with the maximum parsimony or neighbor-joining tree reconstruction method.

Confidence Intervals

The size distribution of insertions and deletions in human and rodent pseudogenes suggests the logarithmic gap penalty for sequence alignment.

The size distributions of deletions, insertions, and indels (i.e., insertions or deletions) were studied, using 78 human processed pseudogenes and other published data sets. The following results were obtained: (1) Deletions occur more frequently than do insertions in sequence evolution; none of the pseudogenes studied shows significantly more insertions than deletions. (2) Empirically, the size distributions of deletions, insertions, and indels can be described well by a power law, i.e., fk = Ck-b, where fk is the frequency of deletion, insertion, or indel with gap length k, b is the power parameter, and C is the normalization factor. (3) The estimates of b for deletions and insertions from the same data set are approximately equal to each other, indicating that the size distributions for deletions and insertions are approximately identical. (4) The variation in the estimates of b among various data sets is small, indicating that the effect of local structure exists but only plays a secondary role in the size distribution of deletions and insertions. (5) The linear gap penalty, which is most commonly used in sequence alignment, is not supported by our analysis; rather, the power law for the size distribution of indels suggests that an appropriate gap penalty is wk = a + b ln k, where a is the gap creation cost and blnk is the gap extension cost. (6) The higher frequency of deletion over insertion suggests that the gap creation cost of insertion (ai) should be larger than that of deletion (ad); that is, ai - ad = ln R, where R is the frequency ratio of deletions to insertions.

Animals

Estimating the intensity of male-driven evolution in rodents by using X-linked and Y-linked Ube 1 genes and pseudogenes.

Using sequence data from the last introns of ZFX and ZFY genes, we previously estimated the male-to-female ratio (alpha) of mutation rate to be close to 6 in higher primates and 1.8 in rodents. As the mutation rate may vary among different regions of the mammalian genome, it is interesting to see whether sequence data from other regions will give similar estimates. In this study, we have determined the partial genomic sequences of the ubiquitin-activating enzyme E1 genes (Ube 1x and Ube 1y for the X-linked and Y-linked homologues, respectively) of mice and rats and two mouse Ube 1y pseudogenes. From the intron sequences of the Ube 1 genes, we calculated the divergence of the Y-linked genes (Y = 0.161) and that of the X-linked genes (X = 0.107) between mouse and rat, and found the Y/X ratio to be 1.50. This ratio led to an estimate of alpha = 2.0 with a 95% confidence interval of (1.0, 3.9). Similar estimates of alpha were obtained if mouse Ube 1y pseudogenes were used instead of the mouse Ube 1y functional gene. These estimates are consistent with our previous estimate for rodents and suggest that the sex ratio of mutation rate in rodents is approximately only one-third of that in higher primates. Our estimate of the divergence time between Ube 1x and Ube 1y supports the view that the two genes separated before the eutherian radiation.

Animals

The phylogenetic positions of the conifer genera Amentotaxus Phyllocladus, and Nageia inferred from 18S rRNA sequences.

To determine the evolutionary positions of the conifer genera Amentotaxus, Phyllocladus, and Nageia, we obtained 18S rRNA sequences from 11 new taxa representing the major living orders and families of gymnosperms. With the published Chlamydomonas as an outgroup, phylogenetic analyses of our new data and available sequences indicate that (1) the Gnetales form a monophyletic group, which is an outgroup to the conifers, (2) the conifers are monophyletic, (3) Taxaceae, Cephalotaxaceae, Cupressaceae, and Taxodiaceae form a monophyletic group, (4) Amentotaxus is closer to Torreya than to Cephalotaxus, suggesting that Amentotaxus is better to be classified as a member of Taxaceae, (5) Phyllocladus, Dacrycarpus, Podocarpus, and Nageia form a monophyletic group, and (6) Pinaceae is an out-group to the other families of conifers. Our finding that Phyllocladus is a sister group of the Podocarpaceae disagrees with the suggestion that the phylloclade of the genus is an ancient structure and that the genus is a terminal taxon within the Podocarpaceae. The genus Nageia is more closely related to Podocarpus than to Dacrycarpus and was derived from within the Podocarpaceae. In conclusion, our data indicate that in conifers, the uniovulate cone occurred independently in Taxacaeae and Cephalotaxaceae, and in Podocarpaceae after the three families separated from Pinaceae, and support the hypothesis that the uniovulate cone is derived from reduction of a multiovulate cone.

DNA, Complementary

Maximum likelihood estimation of the heterogeneity of substitution rate among nucleotide sites.

This paper presents a maximum likelihood approach to estimating the variation of substitution rate among nucleotide sites. We assume that the rate varies among sites according to an invariant+gamma distribution, which has two parameters: the gamma parameter alpha and the proportion of invariable sites theta. Theoretical treatments on three, four, and five sequences have been conducted, and computer program have been developed. It is shown that rho = (1 + theta alpha)/(1 + alpha) is a good measure for the rate heterogeneity among sites. Extensive simulations show that (1) if the proportion of invariable sites is negligible, i.e., theta = 0, the gamma parameter alpha can be satisfactorily estimated, even with three sequences; (2) if the proportion of invariable sites is not negligible, the heterogeneity rho can still be suitably estimated with four or more sequences; and (3) the distances estimated by the proposed method are almost unbiased and are robust against violation of the assumption of the invariant + gamma distribution.

Computer Simulation

Effects of MK-447 on thrombin-induced aggregation, secretion of ATP, and [Ca2+]i mobilization in rabbit platelets.

AIM: To study the effects of MK-447 on aggregation release reaction and intracellular calcium mobilization by thrombin. METHODS: Aggregation and release reaction were assessed by light transmission and ATP content in rabbit citrate platelet-rich plasma (PRP), and cytosolic-free calcium was measured by fluorescence and imaging. RESULTS: MK-447 (2-aminomethyl-4-t-butyl-6-iodophenol hydrochloride) induced a decrease in light transmission (DLT), so called platelet shape change, without detectable aggregation and secretion of ATP, and increased intracellular calcium concentration ([Ca2+]i) slightly in washed single platelet loaded with Fura 2, the peak value being about 160 nmol.L-1. These effects were not inhibited by egtazic acid 3 mmol.L-1 or indometacin 3 mumol.L-1. The pretreatment of PRP with MK-447 700 mumol.L-1 reduced the DLT by thrombin, potentiated and enhanced thrombin-induced aggregation and secretion of ATP in a concentration-dependent manner. Thrombin-induced [Ca2+]i mobilization (peak value: 369 +/- 45 nmol.L-1) was further enhanced by the administration of MK-447 at 2 min before the addition of thrombin, and the peak value reached 623 +/- 121 nmol.L-1 (P < 0.01). CONCLUSION: MK-447-induced platelet shape change was involved in intracellular calcium release in this preparation. MK-447 enhanced thrombin-induced aggregation and release reaction and these effects of MK-447 on aggregation and release reaction by thrombin might result from the synergistic effect of intracellular calcium mobilization.

Adenosine Triphosphate

Evoked tensions in rabbit aorta by emptying intracellular Ca2+ stores with cyclopiazonic acid, thapsigargin, and ryanodine.

AIM: To study the increase of plasma membrane Ca2+ permeability in response to depletion of intracellular Ca2+ stores. METHODS: In Ca(2+)-free medium, 2 selective inhibitors of sarcoplasmic reticulum (SR) Ca2+ pump ATPase, cyclopiazonic acid (CPA) and thapsigargin (Tha), and an activator of Ca(2+)-induced Ca2+ release channel (CICR), ryanodine (Rya), depleted intracellular Ca2+ stores sensitive to both caffeine and phenylephrine in rabbit aortic rings and caused sustained tensions when Ca2+ reintroduction. These tensions were taken as the increase of plasma Ca2+ permeability by depletion of intracellular Ca2+ stores. RESULTS: The extracellular Ca(2+)-dependent tensions caused by Tha and Rya 3 mumol.L(-1) and CPA 30 mumol.L(-1) were 0.94, 1.1, and 0.14 g, respectively, and the tension caused by Rya was not inhibited by CPA. CONCLUSION: (a) Besides the depletion of intracellular Ca2+ stores, an activated state of Ca2+ release channels in SR may also mediate the activation of Ca2+ influx from plasma membrane in rabbit aorta; (b) Rya needs caffeine to fully open CICR channel in SR.

Animals

Relationship between platelet density and platelet aggregation, ATP release, and cytosolic-free calcium mobilization in rabbits.

AIM: To study the relationship of platelet density and platelet functions, including aggregation, ATP release, and intracellular calcium mobilization. METHODS: Platelet density was evaluated by discontinuous gradients of Percoll and cytosolic-free calcium concentration in single platelets was measured by fluorescence and imaging. RESULTS: Rabbit platelets were assorted into 3 subpopulations using discontinuous gradients of Percoll: high density (HD, > 1.062, 20%-30%), intermediate density (ID, around 1.057, 40%-50%) and, low density (LD, < 1.051, 10%-15%). The sizes of platelets in these subpopulations were widely different (HD: 725 +/- 76; ID: 443 +/- 47; and LD: 307 +/- 46) and well correlated with the density (r = 0.988, P < 0.01). Thrombin 0.5 U-induced aggregations in the 3 subpopulations were 78 +/- 8%, 69 +/- 4%, and 62 +/- 3%, respectively and a difference was found between HD and LD (P < 0.05). The amounts of ATP released during aggregation were 4.7 +/- 0.9, 3.4 +/- 1.0, and 2.6 +/- 0.6 mumol/4 x 10(5) platelets in HD, ID, and LD groups, respectively. Thrombin 150 U-induced [Ca2+]i mobilization in HD (990 +/- 130 nmol L-1) was different (P < 0.01) from those in ID (410 +/- 80 nmol L-1) and LD (400 +/- 40 nmol L-1). Serotonin (5-HT) 3 mumol L-1 also caused [Ca2+]i mobilization in single-washed platelet subpopulations and results were similar to those of thrombin although it did not induce any detectable aggregation or release reaction. CONCLUSION: Platelet functions and intracellular calcium concentraotion were increased with platelet density in rabbits.

Adenosine Triphosphate

Mouse very-low-density-lipoprotein receptor (VLDLR) cDNA cloning, tissue-specific expression and evolutionary relationship with the low-density-lipoprotein receptor.

The very-low-density-lipoprotein receptor (VLDLR) is a recently described lipoprotein receptor that shows considerable similarity to the low-density-lipoprotein receptor (LDLR). This receptor has been suggested to be important for the metabolism of apoprotein-E-containing triacylglycerol-rich lipoproteins, such as very-low-density-lipoprotein (VLDL), beta-migrating VLDL and intermediate-density lipoprotein. cDNA clones that code for the VLDLR were isolated from a mouse heart cDNA library. The deduced amino acid sequence predicts a mature protein of 846 amino acids preceded by a 27-residue signal peptide. Three mRNA species for the VLDLR with sizes of 3.9, 4.5 and 7.9 kilobases were present in high concentration in heart and muscle, which utilize triacylglycerols as an energy source. VLDLR mRNA is also detected in decreasing amounts in kidney, brain, ovary, testis, lung and adipose tissue. It is essentially absent in liver and small intestine. The amino acid sequence of the VLDLR is highly conserved among rabbit, human and mouse. VLDLR contains five structural domains very similar to those in LDLR, except that the ligand-binding domain in VLDLR has an eightfold repeat instead of a sevenfold repeat in LDLR. Sequence conservation among animal species is much higher for the VLDLR than the LDLR. Sequences of the VLDLR from three vertebrate species and the LDLR from five vertebrate species were aligned and a phylogenetic tree was reconstructed. Although both receptors contain five domains and share amino acid sequence similarity, our computations showed that they diverged before the divergence between mammals and amphibians. In addition, sequence comparison of both receptor sequences suggests that the rabbit is evolutionarily closer to man than to the mouse. These results are consistent with the hypothesis that the VLDLR and the LDLR have evolved from a common ancestral gene to play distinct roles in lipoprotein metabolism and that the metabolic handling of triacylglycerol by the body via the VLDLR is a highly conserved mechanism.

Amino Acid Sequence

Weak male-driven molecular evolution in rodents.

In humans and rodents the male-to-female ratio of mutation rate (alpha m) has been suggested to be extremely large, so that the process of nucleotide substitution is almost completely male-driven. However, our sequence data from the last intron of the X chromosome-linked (Zfx) and Y chromosome-linked (Zfy) zinc finger protein genes suggest that alpha m is only approximately 2 in rodents with a 95% confidence interval from 1 to 3. Moreover, from published data on oogenesis and spermatogenesis we estimate the male-to-female ratio of the number of germ cell divisions per generation to be approximately 2 in rodents, confirming our estimate of alpha m and suggesting that errors in DNA replication are the primary source of mutation. As the estimated alpha m for rodents is only one-third of our previous estimate of approximately 6 for higher primates, there appear to be generation-time effects--i.e., alpha m decreases with decreasing generation time.

Animals

Contrasting rates of nucleotide substitution in the X-linked and Y-linked zinc finger genes.

We have sequenced the entire exon (approximately 1.180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 bp were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans in considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.

Animals

A model for the correlation of mutation rate with GC content and the origin of GC-rich isochores.

Based on the biochemical kinetics of DNA replication and mutagenesis, including misincorporation and correction, a model has been developed for studying the relationships among the mutation rate (u), the G+C content of the sequence (f), and the G+C proportion in the nucleotide precursor pool (N). Also a measure for the next-nucleotide effect, called the maximum capacity of the next-nucleotide effect (MC), has been proposed. Under the normal physiological conditions of mammalian germ cells, our results indicate: (1) the equilibrium G+C content in a sequence is approximately equal to the G+C proportion in the nucleotide precursor pool, i.e., f approximately N, which is independent of the next-nucleotide effect; (2) an inverted-V-shaped distribution of mutation rates with respect to G+C contents is predicted, when the next-nucleotide effect is week, i.e., MC approximately 1; (3) the distribution becomes flatter (i.e., inverted-U-shaped) as MC increases, but the peak at 50% GC is still observed when MC < 2; and (4) the peak disappears when MC > 2.8, that is, when the next-nucleotide effect becomes strong. Our results suggest that changes in the relative concentrations of nucleotide precursors can cause variations among genes both in mutation rate and in G+C content and that compositional isochores (DNA segments with a homogeneous G+C content) can arise in a genome due to differences in replication times of DNA segments.

Base Composition

Intronic gene conversion in the evolution of human X-linked color vision genes.

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.

Animals

Evolution of base composition in the insulin and insulin-like growth factor genes.

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold-blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF-I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.

Animals

[Effects of sodium diethyldithiocarbamate on ischemia-reperfusion-induced brain injury in Mongolian gerbil].

Brain injury in Mongolian gerbil (Merisones unguiculatus) was induced by occluding bilateral common carotid arteries for 60 min followed by reperfusion for 5 or 30 min. Oxygen free radicals in brain tissue were measured by electron spin resonance (ESR) technique, malondialdehyde (MDA) was measured by fluorescence spectrometry, and superoxide dismutase (SOD) was measured by nitrite kit. Oxygen free radicals and MDA were not significantly increased, but activities of T-SOD and Mn-SOD were decreased after 60 min of cerebral ischemia. The free radicals were increased at 5-min reperfusion, and then reduced to the level of ischemia group after 30-min reperfusion. MDA was increased remarkably after reperfusion of 30 min, whereas the activity of SOD continued to decrease. Sodium diethyldithiocarbamate (DTC), i.v. 5-100 mg.kg-1 15 min before occlusion, decreased the production of MDA and increased the activities of T-SOD and Mn-SOD. The formation of oxygen free radicals was depressed by i.v. DTC 50 mg.kg-1. The result suggested that the protective effects of DTC on ischemia-reperfusion-induced brain injury might be induced by scavenging the oxygen free radicals, increasing the Mn-SOD activity and decreasing the production of MDA.

Animals