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Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [ M ( Q 1 )] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all Q >0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95% 3 )] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P >0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95% CI NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.CI ) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

SARM1, an executioner in axon degeneration, is an autoinhibitory NAD-consuming enzyme, composed of multiple domains. NMN and its analogs, CZ-48 and VMN, are the only known activators, which can release the inhibitory ARM domain from the enzymatic TIR domain. Here, we document that acid can also activate SARM1, even more efficiently than NMN, possibly via the protonation of the negative residues. Systematic mutagenesis revealed that a single mutation, E689Q in TIR, led to the constitutive activation of SARM1. It forms a salt bridge with R216 in the neighboring ARM, maintaining the autoinhibitory structure. Using this 'acid activation' protocol, mutation K597E was found to inhibit activation, while H685A eliminated SARM1 catalytic activity, revealing two distinct inhibitory mechanisms. The protocol has also been applied to differentiate two classes of chemical inhibitors. NAD, dHNN, disulfiram, CHAPS, and TRX-100 mainly inhibited the activation process, while nicotinamide and Tweens mainly inhibited SARM1 catalysis. Taken together, we demonstrate a new mechanism for SARM1 activation and decipher two distinct inhibitory mechanisms of SARM1., (© 2021 Federation of European Biochemical Societies.)

Objective: To identify imaging markers that reflect the epidermal growth factor receptor (EGFR) mutation status by comparing computed tomography (CT) imaging-based histogram features between bone metastases with and without EGFR mutation in patients with primary lung adenocarcinoma., Materials and Methods: This retrospective study included 57 patients, with pathologically confirmed bone metastasis of primary lung adenocarcinoma. EGFR mutation status of bone metastases was confirmed by gene detection. The CT imaging of the metastatic bone lesions which were obtained between June 2014 and December 2017 were collected and analyzed. A total of 42 CT imaging-based histogram features were automatically extracted. Feature selection was conducted using Student's t-test, Mann-Whitney U test, single-factor logistic regression analysis and Spearman correlation analysis. A receiver operating characteristic (ROC) curve was plotted to compare the effectiveness of features in distinguishing between EGFR(+) and EGFR(-) groups. DeLong's test was used to analyze the differences between the area under the curve (AUC) values., Results: Three histogram features, namely range, skewness, and quantile 0.975 were significantly associated with EGFR mutation status. After combining these three features and combining range and skewness, we obtained the same AUC values, sensitivity and specificity. Meanwhile, the highest AUC value was achieved (AUC 0.783), which also had a higher sensitivity (0.708) and specificity (0.788). The differences between AUC values of the three features and their various combinations were statistically insignificant., Conclusion: CT imaging-based histogram features of bone metastases with and without EGFR mutation in patients with primary lung adenocarcinoma were identified, and they may contribute to diagnosis and prediction of EGFR mutation status.

Purpose: To better understand the gene mutational status and heterogeneity between primary and metastatic CRC (mCRC) using a sensitive sequencing method., Methods: The mutational status of EGFR, KRAS, NRAS, PIK3CA, ERBB2, BRAF, KIT, and PDGFRA was analyzed in 65 patients, with 147 samples of primary and paired live or lung metastatic CRC, using next-generation sequencing (NGS), quantitative RT-PCR (qPCR), and Sanger sequencing., Results: Fifteen cases (15/22, 68.2%) of lung mCRC and thirteen cases (13/20, 65%) of liver mCRC harboured the same mutation profiles of KRAS, NRAS, or BRAF in the primary lesions. To all detected genes, 11 cases (11/22, 50%) of lung mCRC and 11 cases (11/20, 55%) of liver mCRC showed different mutational genes in the primary tumours. KRAS and BRAF mutations were more frequent in lung metastatic lesions (p = 0.004 and 0.003, respectively). The gene mutations in KRAS, NRAS, BRAF, and PIK3CA in the lung metastatic sites were more frequent than those in the liver metastatic sites (86.7 vs. 44%, respectively, p = 0.000). Some new mutations were not covered in the qPCR ranges but were detected by NGS., Conclusion: The study demonstrated that the discordance of gene mutational status between paired primary and metastatic tumours is rather high when detected by NGS. Evaluating the mutational status of both the primary and metastatic tumours should be considered in clinical mutation testing.

Thermotolerance is a polygenic trait that contributes to cell survival and growth under unusually high temperatures. Although some genes associated with high-temperature growth (Htg+) have been identified, how cells accumulate mutations to achieve prolonged thermotolerance is still mysterious. Here, we conducted experimental evolution of a Saccharomyces cerevisiae laboratory strain with stepwise temperature increases for it to grow at 42 °C. Whole genome resequencing of 14 evolved strains and the parental strain revealed a total of 153 mutations in the evolved strains, including single nucleotide variants, small INDELs, and segmental duplication/deletion events. Some mutations persisted from an intermediate temperature to 42 °C, so they might be Htg+ mutations. Functional categorization of mutations revealed enrichment of exonic mutations in the SWI/SNF complex and F-type ATPase, pointing to their involvement in high-temperature tolerance. In addition, multiple mutations were found in a general stress-associated signal transduction network consisting of Hog1 mediated pathway, RAS-cAMP pathway, and Rho1-Pkc1 mediated cell wall integrity pathway, implying that cells can achieve Htg+ partly through modifying existing stress regulatory mechanisms. Using pooled segregant analysis of five Htg+ phenotype-orientated pools, we inferred causative mutations for growth at 42 °C and identified those mutations with stronger impacts on the phenotype. Finally, we experimentally validated a number of the candidate Htg+ mutations. This study increased our understanding of the genetic basis of yeast tolerance to high temperature.

Objective: To determine the gene location of two Gitelman syndrome (GS) family SLC12A3 genes and explore treatments using Chinese medicine (CM) prescriptions., Methods: In order to locate the two GS mutations, samples were collected from 11 people from two different pedigrees for direct genetic sequencing and comparison of the 26 exons of SLC12A3. Furthermore, the change of serum potassium was monitored throughout the therapy and those two probands undertook a sequential superposition of Western medicine (including potassium, Panangin and potassium-sparing diuretics) with CM prescription based on Buyang Huanwu Decoction () and Sijunzi Decoction (). The treatment included three stages, oral potassium chloride for the first 2 weeks (stage 1), potassium-sparing diuretic and Panangin with potassium chloride for the next 2 weeks (stage 2), CM along with the medicine in stage 2 for the final 2 weeks (stage 3)., Results: The three mutations occurring in proband 1 from pedigree I were Thr60Met, 965-1_976del13ins12 (small indels mutation) and Ala122Ala (homozygous silent mutation). Likewise, three mutations, Asn359Lys, Thr382Met and Arg913Gln, appeared in the proband 2 from pedigree II. The serum potassium levels increasing from baseline to sequential stages were 1.63 mmol/L (baseline), 2.5 mmol/L (stage 1), 3.1 mmol/L (stage 2) and 3.9 mmol/L (stage 3) in the proband 1, and 2.8 mmol/L (baseline), 3.1 mmol/L (stage 1), 3.5 mmol/L (stage 2) and 4.3 mmol/L (stage 3) in the proband 2, respectively. The symptoms (numbness of limbs, weakness, palpitations, etc.) of both probands were all alleviated., Conclusions: The mutations of both GS pedigrees can be defined as compound heterozygous mutations, most of which are known as missense mutations. Applying CM could be an appropriate choice for future intervention of GS.

Oral squamous cell carcinoma (OSCC), an epithelial malignancy affecting a variety of subsites in the oral cavity, is prevalent in Asia. The survival rate of OSCC patients has not improved over the past decades due to its heterogeneous etiology, genetic aberrations, and treatment outcomes. Improvement in therapeutic strategies and tailored treatment options is an unmet need. To unveil the mutational spectrum, whole-exome sequencing of 120 OSCC from male individuals in Taiwan was conducted. Analyzing the contributions of the five mutational signatures extracted from the dataset of somatic variations identified four groups of tumors that were significantly associated with demographic and clinical features. In addition, known ( TP53 , FAT1 , EPHA2 , CDKN2A , NOTCH1 , CASP8 , HRAS , RASA1 , and PIK3CA ) and novel ( CHUK and ELAVL1 ) genes that were significantly and frequently mutated in OSCC were discovered. Further analyses of gene alteration status with clinical parameters revealed that the tumors of the tongue were enriched with copy-number alterations in several gene clusters containing CCND1 and MAP4K2 . Through defining the catalog of targetable genomic alterations, 58% of the tumors were found to carry at least one aberrant event potentially targeted by US Food and Drug Administration (FDA)-approved agents. Strikingly, if targeting the p53-cell cycle pathway ( TP53 and CCND1 ) by the drugs studied in phase I-III clinical trials, those possibly actionable tumors are predominantly located in the tongue, suggesting a better prediction of sensitivity to current targeted therapies. Our work revealed molecular OSCC subgroups that reflect etiological and prognostic correlation as well as defined the landscape of major altered events in the coding regions of OSCC genomes. These findings provide clues for the design of clinical trials for targeted therapies and stratification of OSCC patients with differential therapeutic efficacy., Competing Interests: Competing interest: The authors declare that they have no competing interests.

Cancer development and progression result from somatic evolution by an accumulation of genomic alterations. The effects of those alterations on the fitness of somatic cells lead to evolutionary adaptations such as increased cell proliferation, angiogenesis, and altered anticancer drug responses. However, there are few general mathematical models to quantitatively examine how perturbations of a single gene shape subsequent evolution of the cancer genome. In this study, we proposed the gene gravity model to study the evolution of cancer genomes by incorporating the genome-wide transcription and somatic mutation profiles of ~3,000 tumors across 9 cancer types from The Cancer Genome Atlas into a broad gene network. We found that somatic mutations of a cancer driver gene may drive cancer genome evolution by inducing mutations in other genes. This functional consequence is often generated by the combined effect of genetic and epigenetic (e.g., chromatin regulation) alterations. By quantifying cancer genome evolution using the gene gravity model, we identified six putative cancer genes (AHNAK, COL11A1, DDX3X, FAT4, STAG2, and SYNE1). The tumor genomes harboring the nonsynonymous somatic mutations in these genes had a higher mutation density at the genome level compared to the wild-type groups. Furthermore, we provided statistical evidence that hypermutation of cancer driver genes on inactive X chromosomes is a general feature in female cancer genomes. In summary, this study sheds light on the functional consequences and evolutionary characteristics of somatic mutations during tumorigenesis by propelling adaptive cancer genome evolution, which would provide new perspectives for cancer research and therapeutics.

Objective: To determine whether the specific genotype of exon 19 deletion has a better survival outcome than that of exon 21 substitution in advanced lung adenocarcinoma with EGFR mutant patients that were treated with EGFR-TKIs as second-line therapy after first-line chemotherapy., Methods: Between April 1, 2010 and December 31, 2012, the detailed clinical information of 128 patients was screened from the hospital information database of the First Affiliated Hospital and the Third Affiliated Hospital of Kunming Medical University by inclusion/exclusion criteria. Then, a telephone follow-up and a review of all patients' image data were done to obtain the survival information of all patients. After that, all patients' data were processed by IBM(®) SPSS(®) version 19.0., Results: There were correlations between EGFR mutation status, gross tumor type and PFS or OS according to the Kaplan-Meier survival analyses and log-rank tests. The exon 19 deletions had significantly better survival outcomes in comparison to exon 21 substitutions (median PFS: 8.1 vs. 6.8 months, P = 0.002; median OS: 17.6 vs. 12.5 months, P = 0.000). Stratification analyses of PFS and OS revealed that exon 19 deletions had a survival superior to exon 21 substitutions., Conclusion: Compared with L858R mutation, the genotype of exon 19 deletion had a better survival outcome in terms of PFS and OS in patients with advanced lung adenocarcinoma treated with EGFR-TKIs as second-line therapy after first-line chemotherapy.

The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7(C703I/H)) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E,17E,21-tetraen-3β-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17α/β exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair-chair-chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7(C703X) site-saturated mutations or the ERG7(F699T/C703I) double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Cancer cells evolve from normal cells by somatic mutations and natural selection. Comparing the evolution of cancer cells and that of organisms can elucidate the genetic basis of cancer. Here we analyse somatic mutations in >400 cancer genomes. We find that the frequency of somatic single-nucleotide variations increases with replication time during the S phase much more drastically than germ-line single-nucleotide variations and somatic large-scale structural alterations, including amplifications and deletions. The ratio of nonsynonymous to synonymous single-nucleotide variations is higher for cancer cells than for germ-line cells, suggesting weaker purifying selection against somatic mutations. Among genes with recurrent mutations only cancer driver genes show evidence of strong positive selection, and late-replicating regions are depleted of cancer driver genes, although enriched for recurrently mutated genes. These observations show that replication timing has a prominent role in shaping the single-nucleotide variation landscape of cancer cells.

Although targeting the Ras/Raf/MEK pathway remains a promising anticancer strategy, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors in clinical development are likely to be limited in their ability to produce durable clinical responses due to the emergence of acquired drug resistance. To identify potential mechanisms of such resistance, we established MEK inhibitor-resistant clones of human HT-29 colon cancer cells (HT-29R cells) that harbor the B-RafV600E mutation. HT-29R cells were specifically resistant to MEK inhibition in vitro and in vivo, with drug-induced elevation of MEK/ERK and their downstream targets primarily accountable for drug resistance. We identified MEK1(F129L) mutation as a molecular mechanism responsible for MEK/ERK pathway activation. In an isogenic cell system that extended these findings into other cancer cell lines, the MEK1(F129L) mutant exhibited higher intrinsic kinase activity than wild-type MEK1 [MEK1(WT)], leading to potent activation of ERK and downstream targets. The MEK1(F129L) mutation also strengthened binding to c-Raf, suggesting an underlying mechanism of higher intrinsic kinase activity. Notably, the combined use of Raf and MEK inhibitors overcame the observed drug resistance and exhibited greater synergy in HT-29R cells than the drug-sensitive HT-29 parental cells. Overall, our findings suggested that mutations in MEK1 can lead to acquired resistance in patients treated with MEK inhibitors and that a combined inhibition of Raf and MEK may be potentially useful as a strategy to bypass or prevent drug resistance in the clinic.

Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/β-exocyclic side chain stereochemistry.

We compared the single-nucleotide polymorphisms (SNPs) in humans in 182 housekeeping and 148 tissue-specific genes. SNPs were divided into rare and common polymorphisms based on their frequencies. We found that housekeeping genes tend to be less polymorphic than tissue-specific genes for both rare and common SNPs. Using mouse as a second species for computing sequence divergences, we found no evidence of positive selection: for both housekeeping and tissue-specific genes, the ratio of nonsynonymous to synonymous common SNPs per site showed no significant difference from that of divergence. Similarly, we observed no evidence of positive selection for the 289 and 149 genes that have orthologs available for divergence calculation between humans and chimpanzees and between humans and Old World monkeys, respectively. A comparison with previous SNP studies suggests that approximately 20% of the nonsynonymous SNPs in the human population are nearly neutral and that positive selection in the human genome might not be as frequent as previously thought.

Three frequently used methods for estimating the synonymous and nonsynonymous substitution rates (Ks and Ka) were evaluated and compared for their accuracies; these methods are denoted by LWL85, LPB93, and GY94, respectively. For this purpose, we used a codon-evolution model to obtain the expected Ka and Ks values for the above three methods and compared the values with those obtained by the three methods. We also proposed some modifications of LWL85 and LPB93 to increase their accuracies. Our computer simulations under the codon-evolution model showed that for sequences < or =300 codons, the performance of GY94 may not be reliable. For longer sequences, GY94 is more accurate for estimating the Ka/Ks ratio than the modified LPB93 and LWL85 in the majority of the cases studied. This is particularly so when k > or = 3, which is the transition/transversion (mutation) rate ratio. However, when k is approximately 2 and when the sequence divergence is relatively large, the modified LWL85 performed better than GY94 and the modified LPB93. The inferiority of LPB93 to LWL85 is surprising because LPB93 was intended to improve LWL85. Also, it has been thought that the codon-based method of GY94 is better than the heuristic method of LWL85, but our simulation results showed that in many cases, the opposite was true, even though our simulation was based on the codon-evolution model.

It has been hypothesized that the length of an exon tends to increase with the GC content because stop codons are AT-rich and should occur less frequently in GC-rich exons. This prediction assumes that mutation pressure plays a significant role in the occurrence and distribution of stop codons. However, the prediction is applicable not to all exons, but only to the last coding exon of a gene and to single-exon CDS sequences. We classified exons in multiexon genes in eight eukaryotic species into three groups-the first exon, the internal, and the last exon-and computed the Spearman correlation between the exon length and the percentage GC (%GC) for each of the three groups. In only five of the species studied is the correlation for the last coding exon greater than that for the first or internal exons. For the single-exon CDS sequences, the correlation between CDS length and %GC is mostly negative. Thus, eukaryotic genomes do not support the predicted relationship between exon length and %GC. In prokaryotic genomes, CDS length and %GC are positively correlated in each of the 68 completely sequenced prokaryotic genomes in GenBank with genomic GC contents varying from 25 to 68%, except for the wall-less Mycoplasma genitalium and the syphilis pathogen Treponema pallidum. Moreover, the average CDS length and the genomic GC content are also positively correlated. After correcting for genome size, the partial correlation between the average CDS length and the genomic GC content is 0.3217 ( p < 0.025).

The strength of male-driven evolution - that is, the magnitude of the sex ratio of mutation rate - has been a controversial issue, particularly in primates. While earlier studies estimated the male-to-female ratio (alpha) of mutation rate to be about 4-6 in higher primates, two recent studies claimed that alpha is only about 2 in humans. However, a more recent comparison of mutation rates between a noncoding fragment on Y and a homologous region on chromosome 3 gave an estimate of alpha = 5.3, reinstating strong male-driven evolution in hominoids. Several studies investigated variation in mutation rates among genomic regions that may not be related to sex differences and found strong evidence for such variation. The causes for regional variation in mutation rate are not clear but GC content and recombination are two possible causes. Thus, while the strong male-driven evolution in higher primates suggests that errors during DNA replication in the germ cells are the major source of mutation, the contribution of some replication-independent factors such as recombination may also be important.

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.

Human DNA variation is currently a subject of intense research because of its importance for studying human origins, evolution, and demographic history and for association studies of complex diseases. A approximately 10-kb region on chromosome 1, which contains only four small exons (each <155 bp), was sequenced for 61 humans (20 Africans, 20 Asians, and 21 Europeans) and for 1 chimpanzee, 1 gorilla, and 1 orangutan. We found 52 polymorphic sites among the 122 human sequences and 382 variant sites among the human, chimpanzee, gorilla, and orangutan sequences. For the introns sequenced (8,991 bp), the nucleotide diversity (pi) was 0.058% among all sequences, 0.076% among the African sequences, 0.047% among the Asian sequences, and 0.045% among the European sequences. A compilation of data revealed that autosomal regions have, on average, the highest pi value (0.091%), X-linked regions have a somewhat lower pi value (0.079%), and Y-linked regions have a very low pi value (0.008%). The lower polymorphism in the present region may be due to a lower mutation rate and/or selection in the gene containing these introns or in genes linked to this region. The present region and two other 10-kb noncoding regions all show a strong excess of low-frequency variants, indicating a relatively recent population expansion. This region has a low mutation rate, which was estimated to be 0.74 x 10 per nucleotide per year. An average estimate of approximately 12,600 for the long-term effective population size was obtained using various methods; the estimate was not far from the commonly used value of 10,000. Fu and Li's tests rejected the assumption of an equilibrium neutral Wright-Fisher population, largely owing to the high proportion of low-frequency variants. The age of the most recent common ancestor of the sequences in our sample was estimated to be more than 1 Myr. Allowing for some unrealistic assumptions in the model, this estimate would still suggest an age of more than 500,000 years, providing further evidence for a genetic history of humans much more ancient than the emergence of modern humans. The fact that many unique variants exist in Europe and Asia also suggests a fairly long genetic history outside of Africa and argues against a complete replacement of all indigenous populations in Europe and Asia by a small Africa stock. Moreover, the ancient genetic history of humans indicates no severe bottleneck during the evolution of humans in the last half million years; otherwise, much of the ancient genetic history would have been lost during a severe bottleneck. We suggest that both the "Out of Africa" and the multiregional models are too simple to explain the evolution of modern humans.

The relationship between change in genomic GC content and protein evolution in bacteria was studied by simple correlational analysis (at the genus level) and by Felsenstein's (1985) independent contrast test. We first used the dnaA gene in bacteria as an example to show (1) that the amino acid composition of a protein can be dramatically affected by mutational pressure (the genomic GC content), (2) that surprisingly, deleting relatively closely-related genera may increase rather than decrease the correlation between genomic GC content and amino acid composition, and (3) that most unexpectedly, as the genomic GC content increases, both strongly hydrophobic and strongly hydrophilic amino acids tend to change to ambivalent amino acids, suggesting that the majority of these amino acid substitutions are not caused by positive Darwinian selection. These patterns were then also shown to hold for the 14 other genes studied, indicating their generality for the evolution of bacterial proteins. As directional mutation pressure can affect the amino acid composition of proteins, it may mislead phylogenetic inference, even if protein instead of DNA sequences are used.

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.

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.

It is commonly believed that the rate of mutation is much higher in males than in females because the number of germ-cell divisions per generation is much larger in males than in females. However, the precise magnitude of the male-to-female mutation rate ratio (alpha m) remains unknown. Recently there have been efforts to estimate alpha m by using DNA sequence data from different species. We have studied the potential problems in such an approach. We found that the rate of synonymous substitution varies about fivefold among X-linked genes, as large as the variation among autosomal genes. This large variation makes the assumption of selective neutrality of synonymous changes dubious, so one should be cautious in using the synonymous rates in X-linked and autosomal genes to estimate alpha m. A similar difficulty was also observed in using nonhomologous intron sequences to estimate alpha m. Contrary to the expectation that X-linked sequences should evolve more slowly than autosomal sequences, the Alu repeat in the last intron of the X-linked zinc finger gene has evolved faster than the four autosomal Alu repeats used in this study. It appears that the best way to estimate alpha m is to use homologous sequences. However, such sequences may be involved in gene conversion events. In fact, we found evidence that the Y-linked and X-linked zinc finger genes have been involved in multiple conversion events during primate evolution. Thus, the possibility of gene conversion should be considered when using homologous sequences to estimate alpha m.

It is commonly believed that the mutation rate is much higher in the human male germ line than in the female germ line because the number of germ-cell divisions per generation is much larger in males than in females. But direct estimation of mutation rates is difficult, relying mainly on sex-linked genetic diseases, so the ratio (alpha m) of male to female mutation rates is not clear. It has been noted that if alpha m is very large, then the rate of synonymous substitution in X-linked genes should be only 2/3 of that in autosomal genes, and comparison of human and rodent genes supported this prediction. As the number of X-linked genes used in the study was small and the X-linked and autosomal sequences were non-homologous, and given that the synonymous rate varies among genes, we sequenced the last intron (approximately 1 kb) of the Y-linked and X-linked zinc-finger-protein genes (ZFY and ZFX) in humans, orang-utans, baboons and squirrel monkeys. The ratio Y/X of the substitution rate in the Y-linked intron to that in the X-linked intron is approximately 2.3, which is close to that estimated from synonymous rates in the ZFY and ZFX genes and implies alpha m approximately 6. This estimate of alpha m supports the view that the evolution of DNA sequences in higher primates is male-driven. It is, however, much lower than the previous estimate and therefore raises a number of issues.

Mutations in the genealogy of the sequences in a random sample from a population can be classified as external and internal. External mutations are mutations that occurred in the external branches and internal mutations are mutations that occurred in the internal branches of the genealogy. Under the assumption of selective neutrality, the expected number of external mutations is equal to theta = 4Ne mu, where Ne is the effective population size and mu is the rate of mutation per gene per generation. Interestingly, this expectation is independent of the sample size. The number of external mutations is likely to deviate from its neutral expectation when there is selection while the number of internal mutations is less affected by the presence of selection. Statistical properties of the numbers of external mutations and of internal mutations are studied and their relationships to two commonly used estimates of theta are derived. From these properties, several new statistical tests based on a random sample of DNA sequences from the population are developed for testing the hypothesis that all mutations at a locus are neutral.

Comparison of plant mitochondrial (mt), chloroplast (cp) and nuclear (n) DNA sequences shows that the silent substitution rate in mtDNA is less than one-third that in cpDNA, which in turn evolves only half as fast as plant nDNA. The slower rate in mtDNA than in cpDNA is probably due to a lower mutation rate. Silent substitution rates in plant and mammalian mtDNAs differ by one or two orders of magnitude, whereas the rates in nDNAs may be similar. In cpDNA, the rate of substitution both at synonymous sites and in noncoding sequences in the inverted repeat is greatly reduced in comparison to single-copy sequences. The rate of cpDNA evolution appears to have slowed in some dicot lineages following the monocot/dicot split, and the slowdown is more conspicuous at nonsynonymous sites than at synonymous sites.

The population dynamics of nearly neutral mutations are studied using a single-site and a multisite model. In the latter model, the nucleotides in a sequence are completely linked and the selection schemes employed are additive, multiplicative, and additive with a threshold. Although the third selection scheme is very different from the first two, the three schemes produce identical results for a wide range of parameter values. Thus the present study provides a general theory for the population dynamics of nearly neutral mutations because the results can also be used to draw inferences about other selection schemes such as stabilizing selection and synergistic selection. It is shown that the number of slightly deleterious mutations accumulated in a sequence can be considerably larger under the multisite model than under the single-site model, particularly if the sequence is long or if the mutation rate per site is high. The results show that even a very slight selective difference between synonymous codons can produce a strong bias in codon usage. Three alternative explanations for the strong bias in codon usage in bacterial and yeast genes are considered. The implications of the present results for molecular evolution are discussed.

In the traditional view of molecular evolution, the rate of point mutation is uniform over the genome of an organism and variation in the rate of nucleotide substitution among DNA regions reflects differential selective constraints. Here we provide evidence for significant variation in mutation rate among regions in the mammalian genome. We show first that substitutions at silent (degenerate) sites in protein-coding genes in mammals seem to be effectively neutral (or nearly so) as they do not occur significantly less frequently than substitutions in pseudogenes. We then show that the rate of silent substitution varies among genes and is correlated with the base composition of genes and their flanking DNA. This implies that the variation in both silent substitution rate and base composition can be attributed to systematic differences in the rate and pattern of mutation over regions of the genome. We propose that the differences arise because mutation patterns vary with the timing of replication of different chromosomal regions in the germline. This hypothesis can account for both the origin of isochores in mammalian genomes and the observation that silent nucleotide substitutions in different mammalian genes do not have the same molecular clock.

The mean and standard deviation of the first arrival time for a single mutant to reach a certain frequency and the mean age of a mutant persisting in a population have been studied using diffusion methods. These quantities are shown to be highly dependent on both the heterozygous effect and the population size. For partially recessive deleterious mutations, both the mean first arrival time and the mean age decrease with increasing selection coefficient against heterozygotes. For overdominant mutations, the mean age always increases very rapidly with increasing heterozygous advantage, while the mean first arrival time first increases rapidly with increasing heterozygous advantage to a maximum and then decreases rapidly with increasing heterozygous advantage. The standard deviation of the first arrival time is small while that of the age is large. The results of this study have been applied to study the case of the sickle cell anemia mutant in Africa. It is argued that the present prevalence may be explained without the necessity of quite so great a heterozygous advantage as .25 or higher as proposed by some workers. A reasonable range for the heterozygous advantage seems to be from .05 to .18.

In order to assess the effect of deleterious mutations on various measures of genic variation, approximate formulas have been developed for the frequency spectrum, the mean number of alleles in a sample, and the mean homozygosity; in some particular cases, exact formulas have been obtained. The assumptions made are that two classes of mutations exist, neutral and deleterious, and that selection is strong enough to keep deleterious alleles in low frequencies, the mode of selection being either genic or recessive. The main findings are: (1) If the expected value (q) of the sum of the frequencies of deleterious alleles is about 10% or less, then the presence of deleterious alleles causes only a minor reduction in the mean number of neutral alleles in a sample, as compared to the case of q = 0. Also, the low- and intermediate-frequency parts of the frequency spectrum of neutral alleles are little affected by the presence of deleterious alleles, though the high-frequency part may be changed drastically. (2) The contribution of deleterious mutations to the expected total number of alleles in a sample can be quite large even if q is only 1 or 2%. (3) The mean homozygosity is roughly equal to (1--2q)/(1 + theta 1), where theta 1 is twice the number of new neutral mutations occurring in each generation in the total population. Thus, deleterious mutations increase the mean heterozygosity by about 2q/(1 + theta 1). The present results have been applied to study the controversial problem of how deleterious mutations may affect the testing of the neutral mutation hypothesis.

Formulas are developed for the distribution of allele frequencies and the mean and variance of heterozygosity under mutation and selection pressures. In large populations, even slight selection drastically changes the shape of the distribution of allele frequencies and reduces heterozygosity. On the other hand, the number of rare alleles in a sample is much less affected by selection. Under genic selection, heterozygosity may decrease with increasing population size. As a test statistic, the variance of heterozygosity can be used to detect the presence of selection, though it is not efficient when selection is very slight.

The basal rate of DNA sequence evolution in enterobacteria, as seen in the extent of divergence between Escherichia coli and Salmonella typhimurium, varies greatly among genes, even when only "silent" sites are considered. The degree of divergence is clearly related to the level of gene expression, reflecting constraints on synonymous codon choice. However, where this constraint is weak, among genes not expressed at high levels, divergence is also related to the chromosomal location of the gene; it appears that genes furthest away from oriC, the origin of replication, have a mutation rate approximately two times that of genes near oriC.

The effect of changes in population size on the correlation between mutation rate and heterozygosity was studied by using two models: sudden change in population size and gradual change. It was shown that the results for the two models are close to each other, unless the rate of change for the latter is exceedingly slow. Thus, in many cases, the former model, which is much simpler than the latter, can be used to treat the present problem. Numerical computations showed that the correlation in a population that is expanding or has expanded in the recent past is stronger while the correlation in a population that is decreasing or has experienced a population reduction or bottleneck in the recent past is weaker than that for an equilibrium population with the same mean heterozygosity. However, regardless of whether the population is at equilibrium or not, the proportion of variation in heterozygosity that is attributable to variation in molecular weight over loci is rather small if the mean heterozygosity of the population is low, say of the order 0.05 or smaller.

We have obtained a revised estimate of the pattern of point mutation by considering more pseudogene sequences. Compared with our previous estimate, it agrees better with expectations based on the double-strand structure of DNA. The revised pattern, like the previous one, indicates that mutation occurs nonrandomly among the four nucleotides. In particular, the proportion of transitional mutations (59%) is almost twice as high as the value (33%) expected under random mutation. The same high proportion of transitions is observed in synonymous substitutions in genes. The proportion of transitional changes observed among electrophoretic variants of human hemoglobin is about the same as that predicted by the revised pattern of mutation. We also show that nonrandom mutation increases, by about 15%, the proportion of synonymous mutations due to single-nucleotide changes in the codon table, and increases, from 10% to 50%, the rate of synonymous mutation in the seven genes studied. However, nonrandom mutation reduces (by about 10%) the proportion of polar changes among nonsynonymous mutations in a gene. As far as single-nucleotide changes (in the codon table) are concerned, nonrandom mutation only slightly favors relatively conservative amino acid interchanges, and has virtually no effect on the proportions of radical changes and nonsense mutations.

A method is developed for simulating the allele frequencies in an equilibrium or transient population under the effects of neutral mutation and random drift. The method is based on diffusion theory and is fast so that it can be used to study in detail the distribution of heterozygosity or any quantity that can be expressed as a function of allele frequencies. It has been applied to study the distribution of heterozygosity and the distribution of the frequencies of the first three most frequent alleles in a population. It also has been applied to study the distribution of the number of alleles shared by two populations that were derived from a common stock.

It is pointed out that the arrival or fixation probability of a mutant gene can be easily inferred analytically. The mean first arrival time for a single overdominant mutation to reach frequency y attains its maximum when x is close to but still slightly less than y/2, where x is the equilibrium frequency of the mutant gene in an infinitely large population. For an advantageous mutation, the mean first arrival time decreases with an increasing degree of dominance if selection is strong, but it first increases, after reaching a maximum, then decreases as the degree of dominance increases, if selection is weak. Contrary to our intuition, the mean age of an advantageous mutant gene increases with increasing degree of dominance, except when selection is very strong. A simple explanation is given in terms of the sojourn time at a particular gene frequency.

Formulae for the arrival probability and first arrival time for a single mutant gene to reach a certain number have been obtained by using a continuous branching process. If the mean of the progeny number of heterozygotes is less than one the arrival probability increases with increasing variance of the progeny distribution whereas if the mean is greater than one the contrary is true. Since most human populations are growing at fairly high rates, the result indicates that the probability for a single mutation to grow to a large number is quite high. Numerical computations show that the mean first arrival time decreases with increasing variance of the progeny distribution of the mutant carriers. The results have been applied to investigate the case of the acheiropodia gene in Brazil. The hypothesis that all acheiropodia genes in Brazil were derived from a single mutation seems to be tenable.