Determination of nucleotide sequences from double-stranded regions of HeLa cell nuclear RNA

Double-stranded regions which comprise about 4% of isolated HeLa cell heterogeneous nuclear RNA have been characterized by RNA fingerprinting and sequencing analysis. The simplicity of the pattern in two-dimensional RNA fingerprints suggests a sequence complexity of about 1000 nucleotides. The nucleotide sequences of six prominent RNase T₁-resistant oligonucleotides (ranging in size from 7 to 9 bases) have been determined using isolated double-stranded nuclear RNA labeled in vivo with ³²P-labeled inorganic phosphate. We conclude that (here exists a substantial subpopulation of simple, potentially complementary sequences common to much of the heterogeneous nuclear RNA population and interspersed with other kinds of sequences.     

Oligopeptide biases in protein sequences and their use in predicting protein coding regions in nucleotide sequences

We have examined oligopeptides with lengths ranging from 2 to 11 residues in protein sequences that show no obvious evolutionary relationship. All sequences in the Protein Identification Resource database were carefully classified by sensitive homology searches into superfamilies to obtain unbiased oligopeptide counts. The results, contrary to previous studies, show clear prejudices in protein sequences. The oligopeptide preferences were used to help decide the significance of sequence homologies and to improve the more general methods for detecting protein coding regions within nucleotide sequences.     

Detecting selection in noncoding regions of nucleotide sequences

We present a maximum-likelihood method for examining the selection pressure and detecting positive selection in noncoding regions using multiple aligned DNA sequences. The rate of substitution in noncoding regions relative to the rate of synonymous substitution in coding regions is modeled by a parameter zeta. When a site in a noncoding region is evolving neutrally zeta = 1, while zeta > 1 indicates the action of positive selection, and zeta < 1 suggests negative selection. Using a combined model for the evolution of noncoding and coding regions, we develop two likelihood-ratio tests for the detection of selection in noncoding regions. Data analysis of both simulated and real viral data is presented. Using the new method we show that positive selection in viruses is acting primarily in protein-coding regions and is rare or absent in noncoding regions.     

Structural attributes of nucleotide sequences in promoter regions of supercoiling-sensitive genes: How to relate microarray expression data with genomic sequences

The level of supercoiling in the chromosome can affect gene expression. To clarify the basis of supercoiling sensitivity, we analyzed the structural features of nucleotide sequences in the vicinity of promoters for the genes with expression enhanced and decreased in response to loss of chromosomal supercoiling in Escherichia coli. Fourier analysis of promoter sequences for supercoiling-sensitive genes reveals the tendency in selection of sequences with helical periodicities close to 10 nt for relaxation-induced genes and to 11 nt for relaxation-repressed genes. The helical periodicities in the subsets of promoters recognized by RNA polymerase with different sigma factors were also studied. A special procedure was developed for the study of correlations between the intensities of periodicities in promoter sequences and the expression levels of corresponding genes. Significant correlations of expression with the AT content and with AT periodicities about 10, 11, and 50 nt indicate their role in regulation of supercoiling-sensitive genes.     

Repeating sequences in promoter regions of eukaryotic genes]

Promoter regions of eukaryotic genes were analyzed for the presence of repeated fragments. It was found that the promotor sequences are abundant in direct, symmetrical, and inverted repeats. A computer system for searching and visualizing the repeats was developed.     

Discriminant analysis of promoter regions in Escherichia coli sequences

We have previously developed a general method based on the statisticaltechnique of discriminant analysis to predict splice junctionsin eukaryotic mRNA sequences [Nakata, K., Kanehisa, M. and DeLisi,C. (1985) Nucleic Acids Res., 13, 5327–5340]. In orderto evaluate further applicability of this method, we now analyzethe promoter region of Escherichia coli sequences. The attributesused for discrimination include the accuracy of consensus sequencepatterns measured by the perceptron algorithm, the thermal stabilitymap, the base composition and the Calladine-Dickerson rulesfor helical twist angle, roll angle, torsion angle and propellertwist angle. When applied to selected E. coli sequences in theGenBank database, the method correctly identifies 75 % of thetrue promoter regions. Received on May 15, 1987; accepted on April 17, 1988     

Determination of pyrimidine nucleotide sequences of DNA]

A modification of the Burton method for determination of pyrimidine nucleotide blocks (isopliths) of DNA, providing a higher yield of large-sized nucleotide isopliths, is described. The amount of side products (interisopliths) does not exceed their amount upon DNA hydrolysis according to the Burton method. Another advantage of the technique recommended is a considerable shortening of hudrolysis time (20 min instead of 18 hours). The modification described has been successfully used to determine the pyrimidine nucleotide blocks of some warm-blooded animals DNAs. It has been found that the DNA of animals with higher sensitivity to ionised irradiation contains more oligothymidylic sequences as compared to the DNA of animals, less sensitive to irradiation.     

Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes

Hybrid 5' regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF'-'lacZ or nif'-'lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF5' flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the ntrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed in front of the nifD spacer plus promoter from B. japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH'-'lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.     

A genome walking strategy for the identification of nucleotide sequences adjacent to known regions

To identify the transposon insertion sites in a soil actinomycete, Saccharopolyspora spinosa, a genome walking approach, termed SPTA-PCR, was developed. In SPTA-PCR, a simple procedure consisting of TA cloning and a high stringency PCR, following the single primer-mediated, randomly-primed PCR, can eliminate non-target DNA fragments and obtain target fragments specifically. Using SPTA-PCR, the DNA sequence adjacent to the highly conserved region of lectin coding gene in onion plant, Allium chinense, was also cloned.     

The specificity in promoter recognition by the Bacillus subtilis RNA polymerase deltaA and deltaH holoenzymes is connected with periodicities in the promoter nucleotide sequences

Promoters recognized by deltaA-RNA and deltaH-RNA polymerase have different periodic patterns their nucleotide disposition. By using the special method of Fourier analysis for symbolic sequences. Fourier spectra of the primary structure of promoters transcribed bv deltaA-RNA and deltaH-RNA polymerase were obtained. For two data sets, a small one and a big one, a stepwise discriminant analysis with jackknife test based on the spectral characteristics of each of the promoters was performed. It was shown that it is possible to classify the data with the accuracy of 100% sets into classes: promoters that are recognized by the deltaA-RNA polymerise and promoters that are recognized by the deltaH-RNA polymerase. Significant correlations between the promoter strength and the characteristics of their Fourier spectra were obtained. Thus, the periodicity in nucleotide distribution along the polynucleotide chain is the attribute sufficient for promoter recognition by RNA polymerase holoenzyme.     

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.     

The nucleotide sequences at the termini of adenovirus-2 DNA.

The nucleotide sequence of the first 156 residues from the left end and the first 134 residues from the right end of adenovirus-2 DNA have been determined by direct DNA sequencing techniques. The inverted terminal repetition is 102 nucleotide pairs long. The 5′-ends of the intact DNA are resistant to the action of T4 polynucleotide kinase and the 5′ → 3′ exonucleases from phages lambda and T7. This resistance is most likely due to the covalent attachment of the 5′-terminal C residue to the terminal protein. No significant self-complementarity exists within the inverted terminal repetition, making terminal initiation of DNA replication via a self-priming mechanism unlikely. However, the terminal A + T-rich region followed immediately by a very G + C-rich region is consistent with other schemes for adenovirus-2 DNA replication. The left end of adenovirus-2 DNA contains extensive sequence repetition.     

A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill this aim and overcome the time-consuming approach of screening genomic libraries. Usually these protocols rely on specific requirements and strategies, such as the presence of suitable nucleotide restriction sites and ligation of specific single- or double-strand linkers, thus limiting their application to a certain extent. In this paper we present an alternative PCR-based protocol, consisting of four main steps: (i) extension of a sequence-specific primer; (ii) 3'-tailing of extended single-strand DNA; (iii) PCR; and (iv) nested PCR amplifications. This method, which appears to be a valid alternative to the other PCR-based protocols, was used for the identification of sequences flanking the cDNA encoding region of the Lhcb 1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcbl) in the spinach genome.     

Electrostatic potentials of DNA. Comparative analysis of promoter and nonpromoter nucleotide sequences.

Distribution of electrostatic potential of DNA fragments was evaluated. A method for calculation of electrostatic potential distribution based on Coulomb's law is proposed for long DNA fragments (approximately 1000 nucleotide pairs). For short DNA sequences, this technique provides a good correlation with the results obtained using Poisson-Boltzmann equation thus justifying its application in comparative studies for long DNA fragments. Calculation was performed for several DNA fragments from E. coli and bacteriophage T7 genomes containing promoter and nonpromoter regions. The results obtained indicate that coding regions are characterized by more homogeneous distribution of electrostatic potential whereas local inhomogeneity of DNA electrostatic profile is typical for promoter regions. The possible role of electrostatic interactions in RNA polymerase-promoter recognition is discussed.     

Phylogeny of Triticum L. and Aegilops L. genuses inferred from a comparative analysis of nucleotide sequences in promoter rDNA regions of individual species

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat species and aegilopses from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum-aestivum and timopheevi, diploid Aegilops speltoides acted as a maternal form. In addition to plasmatic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilopses confirmed the accepted wheat phylogenies.