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PubMedDate not supplied

[Protection of segments of replicative form I of phiX174 phage DNA recognized by HindII, BspRI, and AluI restrictases by Escherichia coli RNA-polymerase].

The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.

Bacteriophage phi X 174↗
PubMed2001

Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.

Adenosine↗
PubMed1999

Nucleotide analog interference mapping.

Single-atom substitution experiments provide atomic resolution biochemical information concerning RNA structure and function. Traditionally, these experiments are performed using chimeric RNAs generated by reassembly of full-length RNA from a synthetic substituted oligonucleotide and a truncated RNA transcript. Unfortunately, this technique is limited by the technical difficulty of assembling and measuring the effect of each singly substituted molecule in a given RNA. Here we review an alternate method for rapidly screening the effect of chemical group substitutions on RNA function. Nucleotide analog interference mapping is a chemogenetic approach that utilizes a series 5'-O-(1-thio)-nucleoside analog triphosphates to simultaneously, yet individually, probe the contribution of a functional group at every nucleotide position in an RNA molecule. A population of randomly substituted RNAs is prepared by including phosphorothioate-tagged nucleotide analogs in an in vitro transcription reaction. The active molecules in the RNA population are selected by an activity assay, and the location of the analog substitution detrimental to activity is identified by cleavage at the phosphorothioate tag with iodine and resolution of the cleavage fragments by gel electrophoresis. This method, which is as easy as RNA sequencing, is applicable to any RNA that can be transcribed in vitro and has an assayable function. Here we describe protocols for the synthesis of phosphorothioate-tagged analogs and their incorporation into RNA transcripts. The incorporation properties and unique biochemical signatures of each individual analog are discussed.

Adenosine↗
PubMed1990

The sn-1,2-diacylglycerol cholinephosphotransferase of Saccharomyces cerevisiae. Nucleotide sequence, transcriptional mapping, and gene product analysis of the CPT1 gene.

The complete nucleotide sequence of the Saccharomyces cerevisiae CPT1 gene, a structural gene for the sn-1,2-diacylglycerol cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), was determined. The 2,100-nucleotide extent of DNA sequenced contained an open reading frame encoding 407 amino acids interrupted by an intron near its 5'-end. Northern hybridization analysis detected the presence of 1.4- and 1.7-kilobase transcripts corresponding to the CPT1 gene. S1 nuclease mapping experiments indicated that the 1.4-kilobase transcript was initiated 80 nucleotides upstream from the translational start site near a poly(dA-dT) promoter element and established that the predicted intron was removed in vivo. The previously constructed cpt1::LEU2 insertional mutation was shown to involve disruption of the CPT1 open reading frame approximately in the middle; this construct did not support the production of a stable transcript. The CPT1 promoter region contained several elements homologous to the promoter regions of other phospholipid biosynthetic structural genes. A model for the membrane topography of the predicted 46,305-dalton cholinephosphotransferase was constructed on the basis of predictive methods. The presence of seven transmembrane helices and an asymmetric distribution of hydrophilic regions were predicted. Regional protein homologies to the acetylcholine receptor, phosphoglycerate kinase, and several cytidine diphosphate utilizing enzymes suggested a functional asymmetry which precisely correlated with the predicted topological asymmetry.

Amino Acid Sequence↗
PubMed1987

Characterization of two new genes essential for vegetative growth in Saccharomyces cerevisiae: nucleotide sequence determination and chromosome mapping.

Based on nucleotide sequence determination, we have identified two new yeast genes FUN80 and FUN81 located on chromosome XIII. They are both essential for cellular growth but their function is still unknown. FUN80 is closely linked to the ARGRI (or ARG80) gene while FUN81 is located next to the ARGRII (or ARG81) gene. Interestingly, the proteins encoded by these two genes have a long stretch of acidic amino acids within their C-terminal portions.

Amino Acid Sequence↗
PubMed2001

High-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the entire human DiGeorge syndrome critical region 2 (DGCR2) gene at 22q11.2.

We constructed a high-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the DiGeorge syndrome critical region 2 (DGCR2) gene at chromosome 22q11.2, a human counterpart of mouse seizure-related gene SEZ-12. A total of 102 SNPs were isolated from the region by systematic screening among 48 Japanese individuals: 9 SNPs in the 5' flanking region, 3 in the 5' untranslated region, 2 in the coding regions, 77 in introns, 7 in the 3' untranslated region, and 4 in the 3' flanking region. By a comparison of our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, 80 SNPs (78.4%) were considered to be novel. The ratio of transition to transversion was 3.08:1. In addition, eight other types of genetic variations (one GA dinucleotide polymorphism and seven insertion/deletion polymorphisms) were discovered. The high-resolution map that we constructed will be a useful resource for analyzing gene scans of complex diseases mapped to this local segment on chromosome 22.

3' Untranslated Regions↗
PubMed1988

The gene for the alpha i1 subunit of human guanine nucleotide binding protein maps near the cystic fibrosis locus.

The gene for the alpha i1 subunit of human guanine nucleotide binding (G) protein was mapped by in situ hybridization to chromosome 7 at band q21. The regional chromosomal location of the human alpha i1 gene was confirmed using human/mouse somatic-cell hybrid lines containing portions of human chromosome 7. Because the alpha i1 gene mapped near the cystic fibrosis locus and because an abnormal G protein might be expected to contribute to the pathophysiology of this disease, the alpha i1 gene was mapped with respect to the cystic fibrosis locus as defined by the Met oncogene and anonymous DNA marker pJ3.11. The location of the alpha i1 gene proved to be distinct from that of the cystic fibrosis locus.

Chromosome Banding↗
PubMed1999

Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.

The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.

Base Sequence↗
PubMed2006

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an F1 (p50T x C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

Animals↗
PubMed1997

Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping.

Improved atomic resolution biochemical methods are needed to identify the chemical groups within an RNA that are essential to its activity. As a step toward this goal, we report the use of 5'-O-(1-thio)inosine monophosphate (IMP alphaS) in a nucleotide analog interference mapping (NAIM) assay that makes it possible to simultaneously, yet individually, determine the contribution of almost every N2 exocyclic amine of G within a large RNA. Using IMP alphaS, we identified the exocyclic amines that are essential for 5' or 3' exon ligation by the Tetrahymena group I intron. We report that the amino groups of three phylogenetically conserved guanosines (G111, G112, and G303) are important for 3' exon ligation. The amine of G22, as well as the amines of the other four guanosines within the P1 helix, are essential for ligation of the 5' exon. Previous work has shown that point mutation of either G22 or G303 to an adenosine (A) substantially reduces activity. Like inosine, adenosine lacks an N2 amino group. Interference rescue of the G22A and G303A point mutations was detected at the site of mutation by NAIM using 5'-O-(1-thio)diaminopurine riboside monophosphate (DMP alphaS), an adenosine analog that has an N2 exocyclic amine. The G22A point mutant could also be rescued by incorporation of DMP alphaS at A24. By analogy to genetics, there are interference phenotypes comparable to loss of function, reversion, and suppression. This method can be readily extended to other nucleotide analogs for the analysis of chemical groups essential to a variety of RNA and DNA activities.

Animals↗
PubMed2003

A 3.9-centimorgan-resolution human single-nucleotide polymorphism linkage map and screening set.

Recent advances in technologies for high-throughout single-nucleotide polymorphism (SNP)-based genotyping have improved efficiency and cost so that it is now becoming reasonable to consider the use of SNPs for genomewide linkage analysis. However, a suitable screening set of SNPs and a corresponding linkage map have yet to be described. The SNP maps described here fill this void and provide a resource for fast genome scanning for disease genes. We have evaluated 6,297 SNPs in a diversity panel composed of European Americans, African Americans, and Asians. The markers were assessed for assay robustness, suitable allele frequencies, and informativeness of multi-SNP clusters. Individuals from 56 Centre d'Etude du Polymorphisme Humain pedigrees, with >770 potentially informative meioses altogether, were genotyped with a subset of 2,988 SNPs, for map construction. Extensive genotyping-error analysis was performed, and the resulting SNP linkage map has an average map resolution of 3.9 cM, with map positions containing either a single SNP or several tightly linked SNPs. The order of markers on this map compares favorably with several other linkage and physical maps. We compared map distances between the SNP linkage map and the interpolated SNP linkage map constructed by the deCode Genetics group. We also evaluated cM/Mb distance ratios in females and males, along each chromosome, showing broadly defined regions of increased and decreased rates of recombination. Evaluations indicate that this SNP screening set is more informative than the Marshfield Clinic's commonly used microsatellite-based screening set.

Alleles↗
PubMed2003

Quantitation of free energy profiles in RNA-ligand interactions by nucleotide analog interference mapping.

RNA interactions with protein and small molecule ligands serve a wide variety of biochemical functions in the cell. To best understand the specificity and affinity of these interactions, the free energy contribution made by individual function groups in the RNA must be determined. As an efficient method for obtaining such energetic profiles, we report quantitative nucleotide analog interference mapping (QNAIM). This extension of the NAIM methodology uses the magnitude of analog interference as a function of ligand concentration to calculate binding constants for RNA with individual analog substitutions. In this way, QNAIM not only defines which functional groups are important to an interaction but simultaneously determines the energetic contribution made by each occurrence of that functional group within the RNA polymer. To establish the utility of this approach, QNAIM was used to quantify functional group interactions within the signal recognition particle (SRP), specifically the 4.5S RNA with the M domain of Ffh. In each of the cases in which energetic data were available from previous site-specific substitution analyses, QNAIM provided nearly equivalent results. These experiments on a model system demonstrate that QNAIM is an efficient method to establish a chemically detailed free energy profile for a wide variety of RNA-ligand interactions.

Base Pairing↗
PubMed2006

A single nucleotide polymorphism fine mapping study of chromosome 1q42.1 reveals the vulnerability genes for schizophrenia, GNPAT and DISC1: Association with impairment of sustained attention.

BACKGROUND: The marker D1S251 of chromosome 1q42.1 showed significant association with schizophrenia in a Taiwanese sample. We used single nucleotide polymorphism (SNP) fine mapping to search for the vulnerability genes of schizophrenia. METHODS: We selected 120 SNPs covering 1 Mb around D1S251 from the public database. These selected SNPs were initially validated if allele frequency was >10%. Forty-seven validated SNPs were genotyped in 102 families with at least 2 siblings affected with schizophrenia. RESULTS: Two SNP blocks showed significant association with schizophrenia. Block 1 (five-SNP), located between intron 2 and intron 13 of the glyceronephosphate O-acyltransferase (GNPAT) gene, showed the most significant associations using single-locus TDT (z = -2.07, p = .038, df = 1) and haplotype association analyses (z = -1.99, p = .046, df = 1). Block 2 (two-SNP), located between intron 4 and intron 5 of the disrupted-in-schizophrenia 1 (DISC1) gene, also showed the most significant results in both the single-locus (z = -3.22, p = .0013, df = 1) and haplotype association analyses (z = 3.35, p = .0008, df = 1). The association of the DISC1 gene with schizophrenia was mainly in the patient group with sustained attention deficits as assessed by the Continuous Performance Test. CONCLUSIONS: Chromosome 1q42.1 harbors GNPAT and DISC1 as candidate genes for schizophrenia, and DISC1 is associated with sustained attention deficits.

Acyltransferases↗
PubMed2002

pK(a) perturbation in genomic Hepatitis Delta Virus ribozyme catalysis evidenced by nucleotide analogue interference mapping.

The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pK(a) perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs, in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.

Base Sequence↗
PubMed2006

A high-resolution single nucleotide polymorphism genetic map of the mouse genome.

High-resolution genetic maps are required for mapping complex traits and for the study of recombination. We report the highest density genetic map yet created for any organism, except humans. Using more than 10,000 single nucleotide polymorphisms evenly spaced across the mouse genome, we have constructed genetic maps for both outbred and inbred mice, and separately for males and females. Recombination rates are highly correlated in outbred and inbred mice, but show relatively low correlation between males and females. Differences between male and female recombination maps and the sequence features associated with recombination are strikingly similar to those observed in humans. Genetic maps are available from http://gscan.well.ox.ac.uk/#genetic_map and as supporting information to this publication.

Animals↗
PubMed2005

Effect of genotyping error in model-free linkage analysis using microsatellite or single-nucleotide polymorphism marker maps.

Errors while genotyping are inevitable and can reduce the power to detect linkage. However, does genotyping error have the same impact on linkage results for single-nucleotide polymorphism (SNP) and microsatellite (MS) marker maps? To evaluate this question we detected genotyping errors that are consistent with Mendelian inheritance using large changes in multipoint identity-by-descent sharing in neighboring markers. Only a small fraction of Mendelian consistent errors were detectable (e.g., 18% of MS and 2.4% of SNP genotyping errors). More SNP genotyping errors are Mendelian consistent compared to MS genotyping errors, so genotyping error may have a greater impact on linkage results using SNP marker maps. We also evaluated the effect of genotyping error on the power and type I error rate using simulated nuclear families with missing parents under 0, 0.14, and 2.8% genotyping error rates. In the presence of genotyping error, we found that the power to detect a true linkage signal was greater for SNP (75%) than MS (67%) marker maps, although there were also slightly more false-positive signals using SNP marker maps (5 compared with 3 for MS). Finally, we evaluated the usefulness of accounting for genotyping error in the SNP data using a likelihood-based approach, which restores some of the power that is lost when genotyping error is introduced.

Chromosome Mapping↗
PubMed1985

The RAD2 gene of Saccharomyces cerevisiae: nucleotide sequence and transcript mapping.

We have determined the nucleotide (nt) sequence of a segment of the yeast chromosome carrying the RAD2 gene. The coding region consists of 2925 bp and could encode a protein of 975 amino acids with a calculated Mr of 111 100. A major transcriptional start point was mapped to a position of approx. 22 bp upstream from the first ATG codon. A number of minor transcriptional start points were also identified in this region, all of them 5' to the putative translational start codon. We noted a number of consensus nt sequences in the 5' and 3' non-coding regions of the RAD2, RAD1 and RAD3 genes of Saccharomyces cerevisiae. In addition, three regions of amino acid sequence homology in the putative RAD1 and RAD2 polypeptides were observed.

Amino Acid Sequence↗
PubMed2006

High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations.

Kinesin is an ATP-driven microtubule (MT)-based motor fundamental to organelle transport. Although a number of kinesin crystal structures have been solved, the structural evidence for coupling between the bound nucleotide and the conformation of kinesin is elusive. In addition, the structural basis of the MT-induced ATPase activity of kinesin is not clear because of the absence of the MT in the structure. Here, we report cryo-electron microscopy structures of the monomeric kinesin KIF1A-MT complex in two nucleotide states at about 10 A resolution, sufficient to reveal the secondary structure. These high-resolution maps visualized clear structural changes that suggest a mechanical pathway from the nucleotide to the neck linker via the motor core rotation. In addition, new nucleotide binding pocket conformations are observed that are different from X-ray crystallographic structures; it is closed in the 5'-adenylyl-imidodiphosphate state, but open in the ADP state. These results suggest a structural model of biased diffusion movement of monomeric kinesin motor.

Binding Sites↗