Characteristics of nucleotide blocks in coding and non-coding DNA sequences from different organisms

A statistical analysis of occurrence of particular nucleotide runs (1 divided by 10 nucleotides long) in DNA sequences of different species has been carried out. There are considerable differences in run distributions in DNA sequences of prokaryotes, invertebrates and vertebrates. Distribution of various types of runs has been found to be different in coding and non-coding sequences. There is an abundance of short runs 1 divided by 2 nucleotides long in coding sequences, and there is a deficiency of such runs in the non-coding regions. However, some interesting exceptions from this rule exist: for run distribution of adenine in prokaryotes and for distribution of purine-pyrimidine runs in eukaryotes. This may be stipulated by the fact that the distribution of runs are predetermined by structural peculiarities of the entire DNA molecule. Runs of guanine or cytosine of three to six nucleotides long occur predominantly in the non-coding DNA regions in eukaryotes, especially in vertebrates.     

Estimating the total number of nucleotide substitutions since the common ancestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Summary The mRNA sequences of beta hemoglobin for human, mouse and rabbit were examined. Observations included the following: (1) there is a significant bias against the use of codons only one nucleotide different from terminating codons; (2) less than 4% of the codons end in adenine; (3), guanine is the most common third position nucleotide but it never follows a second position cytosine; (4) nearest neighbor (doublet) nucleotides are non-random with the greatest contributor to non-randomness being the third position suggesting that codon choice for a given amino acid rather than a choice among amino acids is the more important contributor; (5) the CG dinucleotide is even rarer in positions other than the first and second of the codon than it is in those two, suggesting that the need for arginine has in fact elevated the CG frequency in those positions; (6) 77 per cent of the nucleotides are unsubstituted among these three taxa, which could be a sampling effect, but there is strong evidence that about one-third of them are in fact unsubstitutable because of selective constrainsts; (7) the two longest stretches of unsubstituted nucleotides (32 and 35 consecutive nucleotides) surround the points of the two non-coding insertion sequences; (8) over half the substitutions occur in the third nucleotide position of the codons; (9) silent (non-amino acid changing) substitutions occur at about four times the rate of non-silent substitutions on the basis of their relative opportunity to occur; (10) silent substitutions occur slightly but significantly more often in codons that also have non-silent substitutions than independence of the two events would predict; (11) substitutions occur in adjacent nucleotides significantly more often than chance would predict; (12) among four-fold degenerate codons, third position transitions (principally cytosine-uracil interchanges) outnumber transversions by two to one although the reverse ratio would be expected.The analysis of these messengers provided an opportunity to evaluate the random evolutionary hit (REH) theory. I observed that: (1) the REH theory is premised upon five assumptions, all false; (2) the theory leads to contradictory estimates of the number of varions; (3) the REH values are underestimates; (4) the REH values frequently violate the triangle inequality; (5) the REH values, contrary to claim, are not concordant either with accepted point mutations (PAMs) or augmented distances; (6) the REH values are more likely than values uncorrected for multiple substitutions to give incorrect phylogenies; and (7) the REH values have statistical problems probably associated with a large variance in its fundamental parameter, r_e. From this I conclude that REH theory is not suitable for its intended purpose of estimating from protein sequences of nucleotide substitutions since the common ancestor of two gene products.     

Nucleotide discrimination with DNA immobilized in the MspA nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.     

Identification of the nucleotide sequences specific for the 5'-flanking regions of the genes regulated by glucocorticoids by a computer analysis method

Nucleotide sequences of 5'-flanking regions of 11 glucocorticoid-regulated genes and 14 genes non-regulated by these hormones were studied using context computer analysis. Consensus TGTTCT, previously found in DNA fragments protected by glucocorticoid-receptor complexes from DNAase I digestion, was shown to be nonspecific for glucocorticoid-regulated genes. However, the analysis of sequences flanking the TGTTCT consensus has revealed that only glucocorticoid-regulated genes contain four regularly distributed cytosine residues, one of them belonging to TGTTCT consensus. Three of cytosine residues are separated by 8-10 bp, which provides their close neighbourhood at one side of DNA double helix; the fourth extreme cytosine residue is located 6 bp from the nearest one and therefore, the complementary guanine residue is adjacent to the consensus in DNA helix. It is suggested that the consensus itself and two flanking cytosine and one guanine residues form a specific site for the interaction with glucocorticoid-receptor complex.     

Doublet frequencies in evolutionary distinct groups.

We analyze the dinucleotide frequencies of occurrence and preferences separately within the vertebrates, nonvertebrates, DNA viruses, mitochondria, RNA viruses, bacteria and phage sequences. Over half a million nucleotides from more than 400 sequences were used in this study. Distinct patterns are observed. Some of the patterns are common to all sequences, some to either eukaryotes or prokaryotes and others to the subgroups within them. Doublets are the most basic ingredient of order in nucleotide sequences. We suggest that their preferences and the arrangement of nucleotides in the DNA in general is determined to a large extent by the conformational and packaging considerations of the double helix. Some principles of DNA conformation are viewed in light of our results.     

Statistical analysis of nucleotide runs in coding and noncoding DNA sequences

A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.     

Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy.

Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.     

Analysis of Context Sequence Surrounding Translation Initiation Site from Complete Genome of Model Plants

Regions flanking the translation initiation site (TIS) are thought to play a crucial role in translation efficiency of mRNAs, but their exact sequence and evolution in eukaryotes are still a matter of debate. We investigated the context sequences in 20 nucleotides around the TIS in multi-cellular eukaryotes, with a focus on two model plants and a comparison to human. We identified consensus sequences aaaaaaa(A/G)(A/C)aAUGGcgaataata and ggcggc(g/c)(A/G)(A/C)(G/C)AUGGCggcggcgg for Arabidopsis thaliana and Oryza sativa, respectively. We observe strongly conserved G at position +4 and A or C at position -2; however, the exact nucleotide frequencies vary between the three organisms even at these conserved positions. The frequency of pyrimidines, which are considered sub optimum at position -3, is higher in both plants than in human. Arabidopsis is GC-depleted (AU-enriched) compared to both rice and human, and the enrichment is slightly stronger upstream than downstream of AUG. While both plants are similar though not identical in their variation of nucleotide frequencies, rice and human are more similar to each other than Arabidopsis and human. All three organisms display clear periodicity in A + G and C + U content when analyzing normalized frequencies. These findings suggest that, besides few highly conserved positions, overall structure of the context sequence plays a larger role in TIS recognition than the actual nucleotide frequencies.     

Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.

A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA. Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.     

Evolution of the mitochondrial DNA cytochrome b gene in Tetraonidae birds

Mitochondrial fragments containing the cytochrome b gene (1020 bp in size) of four bird species belonging to four genera of the family Tetraonidae (Tetrao parvirostris, Bonasa umbellus, Lagopus lagopus scoticus, and Falcipennis falcipennis) were directly sequenced. Of the 1020 nucleotide positions, 186 were variable and uniformly distributed over the gene and only 46 were parsimony informative. Most substitutions were synonymous. Replacement substitutions were detected for 15 out of 340 amino acid sites; only four replacements were parsimony informative. The greatest codon bias was found for leucine and serine. The C-T transitions and the G-C transversions were, respectively, the most common (60.7%) and the most rare (5.9%). The mutation frequencies were high at the third codon position (85.2%) and relatively low at the first and the second position. At the third codon position of the species examined, the guanine content was the lowest (3.3%) and the cytosine content was the highest (44.5%). Based on the cytochrome b gene sequences, phylogenetic relationships in the order Galliformes are inferred.     

大黄鱼与小黄鱼细胞色素b基因全序列的比较分析

对大黄鱼、小黄鱼线粒体细胞色素b基因进行了PCR扩增及序列测定,得到1140bp的全序列。大黄鱼和小黄鱼的碱基组成相似,前者T、C、A、G含量分别为28.4%、33.0%、23.2%和15.4%,A+T含量为51.6%;后者T、C、A、G含量分别为26.7%、34.1%、23.8%和15.4%,A+T含量为50.5%。大、小黄鱼cytb基因中三联体密码子中碱基的使用频率很相似,第一位较均一,第二位富含T,第三位富含C。大小黄鱼cytb基因存在明显差异,序列相似性仅为88.95%;两序列间具有126个差异位点;碱基转换/颠换率为3.1,碱基替换多发生在密码子第三位;碱基转换中C\T显著高于A\G,表现出转换偏歧。     

Functionality of mutations at conserved nucleotides in eukaryotic SECIS elements is determined by the identity of a single nonconserved nucleotide.

In eukaryotes, the specific cotranslational insertion of selenocysteine at UGA codons requires the presence of a secondary structural motif in the 3' untranslated region of the selenoprotein mRNA. This selenocysteine insertion sequence (SECIS) element is predicted to form a hairpin and contains three regions of sequence invariance that are thought to interact with a specific protein or proteins. Specificity of RNA-binding protein recognition of cognate RNAs is usually characterized by the ability of the protein to recognize and distinguish between a consensus binding site and sequences containing mutations to highly conserved positions in the consensus sequence. Using a functional assay for the ability of wild-type and mutant SECIS elements to direct cotranslational selenocysteine incorporation, we have investigated the relative contributions of individual invariant nucleotides to SECIS element function. We report the novel finding that, for this consensus RNA motif, mutations at the invariant nucleotides are tolerated to different degrees in different elements, depending on the identity of a single nonconserved nucleotide. Further, we demonstrate that the sequences adjacent to the minimal element, although not required for function, can affect function through their propensity to base pair. These findings shed light on the specific structure these conserved sequences may form within the element. This information is crucial to the design of strategies for the identification of SECIS-binding proteins, and hence the elucidation of the mechanism of selenocysteine incorporation in eukaryotes.     

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.     

Determination of the base composition of deoxyribonucleic acid by measurement of the adenine/guanine ratio

A method is described for determination of the base composition (as guanine+cytosine or adenine+thymine content) of DNA by accurate measurement of the adenine/guanine ratio. The DNA is hydrolysed with 0.03n-hydrochloric acid for 40min. to release the purines. The hydrolysate is subjected to ion-exchange chromatography on Zeo-Karb 225. Apurinic acids are eluted with 0.03n-hydrochloric acid and then guanine and adenine are eluted separately with 2n-hydrochloric acid. Guanine and adenine are each collected as a single fraction, and the amount of base in each case is determined by measuring the volume and the extinction at suitable wavelengths. For use in the calculations, millimolar extinction coefficients in 2n-hydrochloric acid of 12.09 for adenine at 262mmu, and 10.77 for guanine at 248mmu, were determined with authentic samples of bases. The method gives extremely reproducible results: from 12 determinations with calf thymus DNA the adenine/guanine molar ratio had a standard deviation of 0.011; this corresponds to a standard deviation in guanine+cytosine content of 0.2% guanine+cytosine.     

Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein.

Using a binding site selection procedure, we have found that sequence-specific DNA-binding by the mouse c-myb protein involves recognition of nucleotides outside of the previously identified hexanucleotide motif. Oligonucleotides containing a random nucleotide core were immunoprecipitated in association with c-Myb, amplified by the Polymerase Chain Reaction and cloned in plasmids prior to sequencing. By alignment of sequences it was apparent that additional preferences existed at each of three bases immediately 5' of the hexanucleotide consensus, allowing an extension of the preferred binding site to YGRCVGTTR. The contributions of these 5' nucleotides to binding affinity was established in bandshift analyses with oligonucleotides containing single base substitutions; in particular, it was found that replacement of the preferred guanine at position -2 with any other base greatly reduced c-Myb binding. We found that the protein encoded by the related B-myb gene bound the preferred c-Myb site with similar affinity; however, B-Myb and c-Myb showed distinct preferences for the identity of the nucleotide at position -1 relative to the hexanucleotide consensus. This study demonstrates that the c-Myb DNA-binding site is more extensive than recognised hitherto and points to similar but distinct nucleotide preferences in recognition of DNA by related Myb proteins.     

Molecular differences in Adh1-F and Adh1-S alleles in the sugar beet Beta vulgaris L

We compared nucleotide sequences of exon 4 and part of exon 5 of alleles F and S of the Adh1 locus controlling alcohol dehydrogenase in sugar beet. The Adh1-F and Adh1-S sequences of the examined fragment were shown to differ by two nucleotides. Adenine (A) and cytosine (C) of Adh1-F were substituted by respectively thymine (T) and adenine (A) in Adh1-S. Consequently, glutamine and asparagine from the F subunit of ADH1 are replaced by valine and lysine, respectively. Because of differences in the amino acid content, the F subunit is by two elementary charges more negatively charged electrically than the S subunit, which correlates with differences in their electrophoretic mobility. Comparison of the examined Adh1 fragment of sugar beet with its counterparts in other plants showed that the sites bearing substitutions in the former species are classed as variable.