Computational prediction of native protein ligand-binding and enzyme active site sequences

Recent studies reveal that the core sequences of many proteins were nearly optimized for stability by natural evolution. Surface residues, by contrast, are not so optimized, presumably because protein function is mediated through surface interactions with other molecules. Here, we sought to determine the extent to which the sequences of protein ligand-binding and enzyme active sites could be predicted by optimization of scoring functions based on protein ligand-binding affinity rather than structural stability. Optimization of binding affinity under constraints on the folding free energy correctly predicted 83% of amino acid residues (94% similar) in the binding sites of two model receptor-ligand complexes, streptavidin-biotin and glucose-binding protein. To explore the applicability of this methodology to enzymes, we applied an identical algorithm to the active sites of diverse enzymes from the peptidase, beta-gal, and nucleotide synthase families. Although simple optimization of binding affinity reproduced the sequences of some enzyme active sites with high precision, imposition of additional, geometric constraints on side-chain conformations based on the catalytic mechanism was required in other cases. With these modifications, our sequence optimization algorithm correctly predicted 78% of residues from all of the enzymes, with 83% similar to native (90% correct, with 95% similar, excluding residues with high variability in multiple sequence alignments). Furthermore, the conformations of the selected side chains were often correctly predicted within crystallographic error. These findings suggest that simple selection pressures may have played a predominant role in determining the sequences of ligand-binding and active sites in proteins.     

人原发性肝细胞癌中总基因组DNA甲基化变化的研究

目的：研究原发性肝细胞癌组织中总基因组DNA甲基化水平及其与病理学及生物学行为的关系。方法：以甲基化酶温育3H-腺苷甲硫氨酸掺入、液闪计数法分析33例中晚期肝癌手术标本的癌灶和癌旁组织细胞内总基因组DNA甲基化水乎，并以10例正常肝组织作对照比较。结果：肝癌灶内的DNA甲基化水平显著低于癌旁组织（P＜0．05）和正常对照肝组织（P＜0．01），其甲基化水平降低程度与肿瘤的大体形态（多发性或单灶性．结节型或区块型）有关，而与组织学改变（Edmondson分级）、门脉癌栓有否及血清AFP水平无明显关系。结论：人原发性肝细胞癌组织中DNA的甲基化水平有显著降低，值得进一步研究肝细胞癌组织个别癌基因片段甲基化及mRNA表达状况。     

Comparisons of eukaryotic genomic sequences.

A method for assessing genomic similarity based on relative abundances of short oligonucleotides in large DNA samples is introduced. The method requires neither homologous sequences nor prior sequence alignments. The analysis centers on (i) dinucleotide (and tri- and tetra-) relative abundance extremes in genomic sequences, (ii) distances between sequences based on all dinucleotide relative abundance values, and (iii) a multidimensional partial ordering protocol. The emphasis in this paper is on assessments of general relatedness of genomes as distinguished from phylogenetic reconstructions. Our methods demonstrate that the relative abundance distances almost always differ more for genomic interspecific sequence comparisons than for genomic intraspecific sequence comparisons, indicating congruence over different genome sequence samples. The genomic comparisons are generally concordant with accepted phylogenies among vertebrate and among fungal species sequences. Several unexpected relationships between the major groups of metazoa, fungal, and protist DNA emerge, including the following. (i) Schizosaccharomyces pombe and Saccharomyces cerevisiae in dinucleotide relative abundance distances are as similar to each other as human is to bovine. (ii) S. cerevisiae, although substantially far from, is significantly closer to the vertebrates than are the invertebrates (Drosophila melanogaster, Bombyx mori, and Caenorhabditis elegans). This phenomenon may suggest variable evolutionary rates during the metazoan radiations and slower changes in the fungal divergences, and/or a polyphyletic origin of metazoa. (iii) The genomic sequences of D. melanogaster and Trypanosoma brucei are strikingly similar. This DNA similarity might be explained by some molecular adaptation of the parasite to its dipteran (tsetse fly) host, a host-parasite gene transfer hypothesis. Robustness of the methods may be due to a genomic signature of dinucleotide relative abundance values reflecting DNA structures related to dinucleotide stacking energies, constraints of DNA curvature, and mechanisms attendant to replication, repair, and recombination.     

Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences.

A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.     

Relationship of plasma folic acid and status of DNA methylation in human gastric cancer

To evaluate the anti-cancer effects of folic acid at the molecular level, we determined plasma folic acid concentration by radioimmuno-assay and the degree of total genomic DNA methylation by incubating DNA with³H-S-adenosylmethionine (³H-SAM) in the presence of a methylase, and analyzed the methylation status of the c-myc and c-Ha-ras oncogenes by Southern blotting in 21 patients with advanced gastric cancer. The degree of total genomic DNA methylation of cancerous tissues was significantly lower than that of paracancerous and non-cancerous tissues; c-myc and c-Ha-ras oncogenes from cancerous (10/21, 5/10) and paracancerous (13/21, 4/10) tissues were hypomethylated. The plasma folic acid concentration in patients who showed hypomethylation was lower than that patients showing normal methylation. These findings suggest that a decrease in folic acid, and the subsequent DNA hypomethylation, may be involved in human gastric carcinogenesis.     

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/microg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status.     

Isolation and chromosomal localization of unique DNA sequences from a human genomic library.

Recombinant bacteriophage lambda from a human genomic library were screened to indentify human DNA inserts having only unique sequences. Unique human inserts were found in about 1% of the phage screened. One recombinant phage, P3-2, was studied in detail. It contains a human insert of 14.7 kilobases with four internal EcoRI cleavage sites. A restriction map was constructed for EcoRI and BamHI sites. Hybridization of the 32P-labeled P3-2 probe to a Southern blot of EcoRI-digested total human DNA yielded distinct bands at positions corresponding to the human insert fragments contained in P3-2. By using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the human DNA segment in phage P3-2 was assigned to human chromosome 22 by blot hybridization and synteny analysis. In addition, another human DNA segment, 11.4 kilobases, in phage P3-10 was assigned to human chromosome 10 by similar procedures. With this approach, more unique DNA sequences can be isolated, assigned to specific human chromosomes, and used as genetic markers for gene mapping and linkage, polymorphism, and other genetic studies in the human genome.     

Atypical regions in large genomic DNA sequences.

Large genomic DNA sequences contain regions with distinctive patterns of sequence organization. We describe a method using logarithms of probabilities based on seventh-order Markov chains to rapidly identify genomic sequences that do not resemble models of genome organization built from compilations of octanucleotide usage. Data bases have been constructed from Escherichia coli and Saccharomyces cerevisiae DNA sequences of > 1000 nt and human sequences of > 10,000 nt. Atypical genes and clusters of genes have been located in bacteriophage, yeast, and primate DNA sequences. We consider criteria for statistical significance of the results, offer possible explanations for the observed variation in genome organization, and give additional applications of these methods in DNA sequence analysis.     

High-throughput method for analyzing methylation of CpGs in targeted genomic regions

A unique microarray-based method for determining the extent of DNA methylation has been developed. It relies on a selective enrichment of the regions to be assayed by target amplification by capture and ligation (mTACL). The assay is quantitatively accurate, relatively precise, and lends itself to high-throughput determination using nanogram amounts of DNA. The measurements using mTACLs are highly reproducible and in excellent agreement with those obtained by sequencing (r = 0.94). In the present work, the methylation status of >145,000 CpGs from 5,472 promoters in 221 samples was measured. The methylation levels of nearby CpGs are correlated, but the correlation falls off dramatically over several hundred base pairs. In some instances, nearby CpGs have very different levels of methylation. Comparison of normal and tumor samples indicates that in tumors, the promoter regions of genes involved in differentiation and signaling are preferentially hypermethylated, whereas those of housekeeping genes remain hypomethylated. mTACL is a platform for profiling the state of methylation of a large number of CpG in many samples in a cost-effective fashion, and is capable of scaling to much larger numbers of CpGs than those collected here.     

Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis.

Hypermethylation of regulatory sequences at the locus of the pi-class glutathione S-transferase gene GSTP1 was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTP1 expression was found to accompany human prostatic carcinogenesis. Immunohistochemical staining with anti-GSTP1 antibodies failed to detect the enzyme in 88 of 91 prostatic carcinomas analyzed. In vitro, GSTP1 expression was limited to human prostatic cancer cell lines containing GSTP1 alleles with hypomethylated promoter sequences; a human prostatic cancer cell line containing only hypermethylated GSTP1 promoter sequences did not express GSTP1 mRNA or polypeptides. Methylation of cytidine nucleotides in GSTP1 regulatory sequences constitutes the most common genomic alteration yet described for human prostate cancer.     

Patterns of damage in genomic DNA sequences from a Neandertal

High-throughput direct sequencing techniques have recently opened the possibility to sequence genomes from Pleistocene organisms. Here we analyze DNA sequences determined from a Neandertal, a mammoth, and a cave bear. We show that purines are overrepresented at positions adjacent to the breaks in the ancient DNA, suggesting that depurination has contributed to its degradation. We furthermore show that substitutions resulting from miscoding cytosine residues are vastly overrepresented in the DNA sequences and drastically clustered in the ends of the molecules, whereas other substitutions are rare. We present a model where the observed substitution patterns are used to estimate the rate of deamination of cytosine residues in single- and double-stranded portions of the DNA, the length of single-stranded ends, and the frequency of nicks. The results suggest that reliable genome sequences can be obtained from Pleistocene organisms.     

砷化物对HL-60细胞生长及基因组DNA甲基化的影响

目的：研究NaAsO2和 N3AO4cf HL-60细胞生长及基因组DNA甲基化的影响,方法：采用细胞培养和限制性内切酶解分析技术观察分析细胞生长及细胞基因组DNA甲基化水平。结果：（1）中无机砷化物对HL－60细胞生长具有抑制作用,特别是高浓度NaAsO2(2,10umol/L)和Na3AsO4(100,300umol/L)可明显地抑制HL－60细胞的生长（P＜0．01）,随着砷化物浓度的增加,MspI酶解基因组DNA片段的分子量变化不大,但Hpa II酶解片段的分子量逐渐变大,结论：提示基因组DNA中CCGG核苷酸内侧C的甲基化总的水平有上升趋势。