Comparative study of synonymous codon usage variations between the nucleocapsid and spike genes of coronavirus, and C-type lectin domain genes of human and mouse

Coronaviruses (CoVs) are single-stranded RNA viruses which contain the largest RNA genomes, and severe acute respiratory syndrome coronavirus (SARS-CoV), a newly found group 2 CoV, emerged as infectious disease with high mortality rate. In this study, we compared the synonymous codon usage patterns between the nucleocapsid and spike genes of CoVs, and C-type lectin domain (CTLD) genes of human and mouse on the codon basis. Findings indicate that the nucleocapsid genes of CoVs were affected from the synonymous codon usage bias than spike genes, and the CTLDs of human and mouse partially overlapped with the nucleocapsid genes of CoVs. In addition, we observed that CTLDs which showed the similar relative synonymous codon usage (RSCU) patterns with CoVs were commonly derived from the human chromosome 12, and mouse chromosome 6 and 12, suggesting that there might be a specific genomic region or chromosomes which show a more similar synonymous codon usage pattern with viral genes. Our findings contribute to developing the codon-optimization method in DNA vaccines, and further study is needed to determine a specific correlation between the codon usage patterns and the chromosomal locations in higher organisms.     

Epidemiological comparisons of codon usage patterns among HIV-1 isolates from Asia, Europe, Africa and the Americas

To investigate the genomic properties of HIV-1, we collected 3,081 sequences from the HIV Sequence Database. The sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. The relative synonymous codon usage (RSCU) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. The synonymous codon usage patterns based on the geographical regions of African countries showed broad distributions; when all the other regions, including Asia, Europe, and the Americas, were taken into account, the Asian countries tended to be divided into two groups. The sequences were clustered into nine non-CRF subtypes. Among these, subtype C showed the most distinct codon usage pattern. To determine why the codon usage patterns in Asian countries were divided into two groups for four target genes, the sequences of the isolates from the Asian countries were analyzed. As a result, the synonymous codon usage patterns among Asian countries were divided into two groups, the southern Asian countries and the other Asian countries, with subtype 01_AE being the most dominant subtype in southern Asia. In summary, the synonymous codon usage patterns among the individual HIV-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses.     

Fourier Power Spectrum Analysis of Exons for the Period-3 Behavior

The period-3 behaviors of 105 exons from 20 genes in human were studied by Fourier power spectrum. The results indicated that not all exons show the period-3 behavior. The exons were adjusted in order to make them accord with the order of the protein translated, and we found that the period-3 character is relation to the length of exons and the bases distribution in the three codon position. Furthermore, as long as the exons with period-3 behavior accord with the order of protein translated, they would exhibit the synonymous codons usage preference, and the codons with g/c at the third position are used in higher frequency. The results are significant to the gene prediction and the research on the introns.     

Comparative study of codon substitution patterns in foot-and-mouth disease virus (serotype O)

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.     

外显子周期三行为特征的研究

基因中蛋白质的编码区具有周期三行为这一规律已成为目前基因预测的理论基础, 采用功率谱对外显子的周期三行为特征进行了研究, 结果表明: 大多数外显子独立存在于基因中时并不具有周期三行为, 而当基因被剪切后外显子连在一起编码蛋白质的时候才具有周期三行为. 并且这种行为特征与外显子的长度、碱基在密码子三个位置上的分布以及氨基酸密码子的使用偏好均有密切关系, 同时符合蛋白质翻译次序的外显子也具有对密码子使用的偏好性, 这一研究结果对于提高基因预测的准确率以及内含子功能的研究具有重要意义.     

Optimizing doped libraries by using genetic algorithms

The insertion of random sequences into protein-encoding genes in combination with biologicalselection techniques has become a valuable tool in the design of molecules that have usefuland possibly novel properties. By employing highly effective screening protocols, a functionaland unique structure that had not been anticipated can be distinguished among a hugecollection of inactive molecules that together represent all possible amino acid combinations.This technique is severely limited by its restriction to a library of manageable size. Oneapproach for limiting the size of a mutant library relies on doping schemes, where subsetsof amino acids are generated that reveal only certain combinations of amino acids in a proteinsequence. Three mononucleotide mixtures for each codon concerned must be designed, suchthat the resulting codons that are assembled during chemical gene synthesis represent thedesired amino acid mixture on the level of the translated protein. In this paper we present adoping algorithm that reverse translates a desired mixture of certain amino acids into threemixtures of mononucleotides. The algorithm is designed to optimally bias these mixturestowards the codons of choice. This approach combines a genetic algorithm with localoptimization strategies based on the downhill simplex method. Disparate relativerepresentations of all amino acids (and stop codons) within a target set can be generated.Optional weighing factors are employed to emphasize the frequencies of certain amino acidsand their codon usage, and to compensate for reaction rates of different mononucleotidebuilding blocks (synthons) during chemical DNA synthesis. The effect of statistical errors thataccompany an experimental realization of calculated nucleotide mixtures on the generatedmixtures of amino acids is simulated. These simulations show that the robustness of differentoptima with respect to small deviations from calculated values depends on their concomitantfitness. Furthermore, the calculations probe the fitness landscape locally and allow apreliminary assessment of its structure.     

Volatilities of codons and its application in similarity analysis of biological sequences

Volatilities of codons provide us a new way to characterize codons. In this article, we propose a new method measuring volatilities of codons base on the physics–chemical distances between amino acids and mutation frequencies between codons, then by which, we give a new graphical representation scheme for codon sequences. Finally, in order to show the effectiveness of our scheme, we analyze similarity among the coding codon sequences of exon 1 of beta-globin gene of human and those of other 10 species, find the result is consistent with those shown in the literature.     

A computational analysis of molecular evolution for virulence genes of zoonotic novel coronavirus (COVID-19)

Zoonotic Novel coronavirus disease 2019 (COVID-19) is highly pathogenic and transmissible considered as emerging pandemic disease. The virus belongs from a large virus Coronaviridae family affect respiratory tract of animal and human likely originated from bat and homology to SARA-CoV and MERS-CoV. The virus consists of single-stranded positive genomic RNA coated by nucleocapsid protein. The rate of mutation in any virulence gene may influence the phenomenon of host radiation. We have studied the molecular evolution of selected virulence genes (HA, N, RdRP and S) of novel COVID-19. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions. Maximum Likelihood genealogies based on differential gamma distribution rates were used for the analysis of null and alternate hypothesis. The null hypothesis was found more suitable for the analysis using Likelihood Ratio Test (LRT) method, confirming higher rate of substitution. The analysis revealed that RdRP gene had the fastest rate evolution followed by HA gene. We have also reported the new motifs for different virulence genes, which are further useful to design new detection and diagnosis kit for COVID -19.     

Recoding the Genetic Code with Selenocysteine

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site‐specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high‐fidelity sense‐codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl‐tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.     

p53 gene mutations in SKH-1 mouse tumors differentially induced by UVB and combined subcarcinogenic benzo[a]pyrene and UVA

We compared the frequency and spectra of p53 mutations in skin tumors from UVB-irradiated and benzo(a)pyrene-UVA-treated SKH-1 mice. Analysis of p53 mutations using a combination of polymerase chain reaction, denaturing high-performance liquid chromatography, and sequencing shows that the frequency and spectrum of p53 mutations in BaP-UVA-induced tumors are quite different from those in UVB-induced tumors. SKH-1 mice were treated with BaP-UVA or UVB for 30 weeks after which skin tumors were collected for analysis of p53 mutations. Among the 11 BaP-UVA-induced tumors with diameters of 5-10 mm, two displayed mutations in exon 8 yielding a mutation frequency of 18.2%. In contrast, the mutation frequency among BaP-UVA-induced tumors was 10.5%. In UVB-induced tumors, the mutation frequency in exon 8 was highly correlated with tumor size. A total of 77.8% of tumors with diameters larger than 10 mm contained p53 mutations. The overall mutation frequency among UVB-induced tumors was 17.9% in exon 8 and only 3.8% in exon 5. Hotspots for p53 mutation in UVB-induced tumors were found at codons 128 and 149 (exon 5), and at codons 268, 270, 271 and 273-276 (exon 8). In addition to widely recognized C-->T missense mutations, there were also tandem CC-->AG changes coupled with either an insertion of T, a C-->G substitution or G-->C/T mutations. All of the mutations were found at tri- or tetra-pyrimidine sites. Thirty-nine per cent of all p53 mutations occurred at codons 274 and 275; 53% occurred at codons 268-271. Two multiple mutation clusters were located at codons 268-271 and 274-276. Both BaP-UVA and UVB caused C-->T transitions at codon 275 in exon 8. A C-->T mutation at codon 294 was induced only by BaP-UVA treatment. In contrast to UVB treatment, BaP-UVA treatment did not induce any mutations in exon 5. We show that individually subcarcinogenic levels of BaP and UVA synergistically induce a novel p53-mutation fingerprint. This fingerprint could serve as a prognostic indicator for the development of BaP-UVA-induced skin tumors.     

Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

High mutation frequency at Ha-ras exons 1-4 in squamous cell carcinomas from PUVA-treated psoriasis patients

Clinical follow-up studies have revealed that PUVA-treated patients are at increased risk of skin cancer, particularly squamous cell carcinoma (SCC). However, since psoralen and UVA (PUVA) is not only a potent mutagen and carcinogen but also an immunosuppressor, and since other (co)carcinogenic factors often exist in psoriasis patients, the exact causes and mechanisms of PUVA-associated SCC are still not completely understood. In order to fill this gap the tools of molecular epidemiology are being used to study the SCC mutational spectra of p53 and Ha-ras, two of the most commonly mutated genes in human cancers. A previous mutation analysis revealed that SCC in PUVA-treated patients often carried mutated p53 genes and that many of the mutations had the UV fingerprint (i.e. C-->T or CC-->TT transitions at dipyrimidine sites). In the present study DNA-sequencing analysis revealed a total of 18 Ha-ras missense or nonsense mutations at exons 1-4 in 13 of 17 SCC (76%) from 8 of 11 (73%) PUVA-treated psoriasis patients. Six of the 18 mutations (33%) were of UV-fingerprint type (C-->T transitions), five (28%) were at 5'-TpG sites (i.e. potential psoralen-binding sites and thus potentially caused by PUVA) and seven were of other type (39%), including six G:C-->T:A transversions at hotspot codon 12. In addition, in the case of 6 of the 11 subjects (55%) both tumor and normal skin samples contained a T:A-->C:G base change at codon 27 (a 5'-ATT site), a change previously hypothesized to be a possible silent Ha-ras polymorphism at one allele. When we compared the present Ha-ras mutation spectrum with the p53 mutation spectrum from a previous study of the samples, we found that approximately half of the tumors harbored mutations in both Ha-ras and p53. Together, our results indicate that Ha-ras mutations are present in a large proportion of PUVA-associated SCC and that UVB, PUVA and other agents may induce Ha-ras mutations and act together with p53 in the formation of SCC in psoriasis patients.     

SARS病毒核衣壳蛋白的表达与鉴定

依据Genebank中SARS基因组序列和酵母菌对密码子的选择性,采用人工合成的方法,合成了优化的SARS病毒核衣壳蛋白(N)的全基因(1296bp),与CTL表位基因(195bp)重组后,将其克隆到酵母分泌型表达载体pPIC9K中,构建成重组表达载体pPIC9K-N.重组载体转化毕赤酵母GS115,并经MD平板和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115-pPIC9K-N.用甲醇诱导其分泌表达目的蛋白并对表达产物进行分析、浓缩与鉴定.结果表明,SARS病毒核衣壳蛋白能实现在毕赤酵母中高效表达,表达量达到20%,初步纯化后的产物具有良好的抗原特异性.     

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.     

Chemical selectivity of nucleobase adduction relative to in vivo mutation sites on exon 7 fragment of p53 tumor suppressor gene

Damage to p53 tumor suppressor gene is found in half of all human cancers. Databases integrating studies of large numbers of tumors and cancer cell cultures show that mutation sites of specific p53 codons are correlated with specific types of cancers. If the most frequently damaged p53 codons in vivo correlate with the most frequent chemical damage sites in vitro, predictions of organ-specific cancer risks might result. Herein, we describe LC-MS/MS methodology to reveal codons with metabolite-adducted nucleobases by LC-MS/MS for oligonucleotides longer than 20 base pairs. Specifically, we used a known carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) to determine the most frequently adducted nucleobases within codons. We used a known sequence of 32 base pairs (bp) representing part of p53 exon 7 with 5 possible reactive hot spots. This is the first nucleobase reactivity study of a double stranded DNA p53 fragment featuring more than 20 base pairs with multiple reactive sites. We reacted the 32 bp fragment with benzo[a]pyrene metabolite BPDE that undergoes nucleophilic substitution by DNA bases. Liquid chromatography-mass spectrometry (LC-MS/MS) was used for sequencing of oligonucleotide products from the reacted 32 bp fragment after fragmentation by a restriction endonuclease. Analysis of the adducted p53 fragment compared with unreacted fragment revealed guanines of codons 248 and 244 as most frequently targeted, which are also mutated with high frequency in human tumors. Codon 248 is mutated in non-small cell and small cell lung, head and neck, colorectal and skin cancer, while codon 244 is mutated in small cell lung cancer, all of which involve possible BDPE exposure. Results suggest the utility of this approach for screening of adducted p53 gene by drugs and environmental chemicals to predict risks for organ specific cancers.     

A new silent mutation found in the Chinese PAH locus and its role in the prenatal diagnosis of phenylketonuria

A silent mutation or sequence polymorphism. A to T substitution at codon 399 in exon 11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The frequencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09, respectively, based on the analyses of 100 normal individuals and 39 PKU patients using DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagnosis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPs linkage analysis, was prenatally diagnosed with this genetic marker.     

Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis

Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.     

A NEW SILENT MUTATION FOUND IN THE CHINESE PAH LOCUS AND ITS ROLE IN THE PRENATAL DIAGNOSIS OF PHENYLKETONURIA

A silent mutation or sequence polymorphism, A to T substitution at codon 399 in exon11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The fre-quencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09,respectively, based on the analyses of 100 normal individuals and 39 PKU patients usingDNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridizationmethods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagno-sis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPslinkage analysis, was prenatally diagnosed with this genetic marker.