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PubMed1985

Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b.

We prepared antiserum that reacted with a major core polypeptide of approximately 62,000 daltons (62K polypeptide), designated 4b, and its 74K precursor, designated P4b. A cell-free translation product of vaccinia virus late mRNA that comigrated with P4b was specifically immunoprecipitated. The late mRNA encoding P4b hybridized to restriction fragments derived from the left end of the HindIII A fragment and to a lesser extent from the right side of the HindIII D fragment. A polypeptide that comigrated with P4a, the precursor of another major core polypeptide, was synthesized by mRNA that hybridized to DNA segments upstream of the P4b gene. Complete nucleotide sequence analysis of the P4b gene revealed an open reading frame, entirely within the HindIII A fragment, that was sufficient to encode a 644-amino-acid polypeptide of 73K. The 5' end of the P4b mRNA was located at or just above the translational initiation site.

Base Sequence↗
PubMed2000

Linkage mapping and nucleotide polymorphisms of the 6-SFT gene of cool-season grasses.

Fructan plays an important role as an alternate carbohydrate and may contribute to drought and cold-stress tolerances in various plant species. The gene coding for sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10), an enzyme that catalyzes the formation and extension of beta-2,6-linked fructans (levans), is important to fructan synthesis in many cool-season grasses, including cereal species. In this study, we compared a conserved sequence from the 6-SFT gene in barley with comparable sequences in 20 other cool-season grasses. We detected several DNA length polymorphisms, including variations in one simple-sequence repeat (SSR) in a 6-SFT intron of the barley cultivars Steptoe and Morex. Using the 'Steptoe' x 'Morex' doubled-haploid mapping population, the 6-SFT gene was genetically mapped to the distal region in the short arm of barley chromosome 1 (7H), where it is closely linked with trait locus Rpg1. Primers designed from other conserved regions of the barley 6-SFT gene successfully amplified 351- or 354-bp sequences of this gene from diverse cool season grass species. Sequence identities of the PCR products were greater than 80% among the 21 species. Phylogeny, as determined using these DNA sequences, is similar to that obtained from rDNA ITS sequences, and congruent with our current knowledge of genome relationships.

Base Sequence↗
PubMed1989

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).

Amino Acid Sequence↗
PubMed1989

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence.

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.

Amino Acid Sequence↗
PubMed1997

Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts.

The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues. The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D. melanogaster and M. musculus. Despite this, all of the psi residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function. We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H. sapiens.

Animals↗
PubMed1987

Three-dimensional similarity mapping reveals nucleotide sequence relationships among human immunodeficiency virus (HIV) isolates.

A method has been developed to compare and graphically display comparisons between many related nucleotide sequences. Principal coordinate analysis, a multidimensional scaling technique, was used to display DNA homology data in three dimensions. Using these methods, an analysis of the env, gag, and gag/pol overlapping regions of human immunodeficiency virus (AIDS virus) clearly demonstrates a clustering of North American isolates relative to African isolates. Elements of the latter group are dissimilar to one another and to the North America group. For the env region, the North American isolates can be resolved into two distinct groups.

Base Sequence↗
PubMed1982

Structural mapping of nucleotide binding sites on chloroplast coupling factor.

Fluorescence resonance energy transfer was used to measure the distances between three nucleotide binding sites on solubilized chloroplast coupling factor from spinach and between each nucleotide site and two tyrosine residues which are important for catalytic activity. The nucleotide energy donor was 1,N6-ethenoadenosine di- or triphosphate, and the nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine diphosphate. The tyrosine residues were specifically labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazole, which served as an energy acceptor. The results obtained indicate the three nucleotide binding sites form a triangle with sides of 44, 48, and 36 A. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Two of the nucleotide sites are approximately equidistant from each of the two tyrosines: one of the nucleotide sites is about 37 A and the other about 41 A from each tyrosine. The third nucleotide site is about 41 A from one of the tyrosines and greater than or equal to 41 A from the other tyrosine.

Binding Sites↗
PubMed1988

DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.

A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.

Amino Acid Sequence↗
PubMed2002

Single nucleotide polymorphisms and disease gene mapping.

Single nucleotide polymorphisms are the most important and basic form of variation in the genome, and they are responsible for genetic effects that produce susceptibility to most autoimmune diseases. The rapid development of databases containing very large numbers of single nucleotide polymorphisms, and the characterization of haplotypes and patterns of linkage disequilibrium throughout the genome, provide a unique opportunity to advance association strategies in common disease rapidly over the next few years. Only the careful use of these strategies and a clear understanding of their statistical limits will allow novel genetic determinants for many of the common autoimmune diseases to be determined.

Autoimmune Diseases↗
PubMed1998

Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N2-methylguanosine.

Nucleotide analogue interference mapping (NAIM) is a general biochemical method that rapidly identifies the chemical groups important for RNA function. In principle, NAIM can be extended to any nucleotide that can be incorporated into an in vitro transcript by an RNA polymerase. Here we report the synthesis of 5'-O-(1-thio)-N2-methylguanosine triphosphate (m2GalphaS) and its incorporation into two reverse splicing forms of the Tetrahymena group I intron using a mutant form of T7 RNA polymerase. This analogue replaces one proton of the N2 exocyclic amine with a methyl group, but is as stable as guanosine (G) for secondary structure formation. We have identified three sites of m2GalphaS interference within the Tetrahymena intron: G22, G212, and G303. All three of these guanosine residues are known to utilize their exocyclic amino groups to participate in tertiary hydrogen bonds within the ribozyme structure. Unlike the interference pattern with the phosphorothioate of inosine (IalphaS, an analogue that deletes the N2 amine of G), m2GalphaS substitution did not cause interference at positions attributable to secondary structural stability effects. Given that the RNA minor groove is likely to be widely used for helix packing, m2GalphaS provides an especially valuable reagent to identify RNA minor groove tertiary contacts in less well-characterized RNAs.

Animals↗
PubMed2002

The human papillomavirus type 31 late 3' untranslated region contains a complex bipartite negative regulatory element.

The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3' untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF(65), and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element.

3' Untranslated Regions↗
PubMed2006

High-resolution analysis of 9p loss in human cancer cells using single nucleotide polymorphism-based mapping arrays.

Single nucleotide polymorphism (SNP) mapping arrays were used to perform DNA copy number analysis of five human cancer cell lines (four malignant mesotheliomas; one non-small cell lung carcinoma) to identify and map the end-points of deletions of 9p. All five cell lines exhibited homozygous deletions encompassing the CDKN2A (alias INK4A/ARF) and CDKN2B loci. The DNA analysis profiles demarcated precisely two different, but overlapping, deletions in each mesothelioma cell line, but the lung cancer cells showed two copies of a single deletion. In the latter cell line, allele analysis revealed that virtually all SNPs for chromosome 9 were homozygous, suggestive of uniparental disomy. These findings demonstrate the utility of SNP-based mapping arrays for high-resolution analysis of genomic imbalances in cancer cells.

Cell Line, Tumor↗
PubMed1986

Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae.

We determined the nucleotide sequence, mapped the 5' and 3' mRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae. A long open reading frame within the RAD2 transcribed region encodes a protein of 1,031 amino acids with a calculated molecular weight of 117,847. A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function. The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame. The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame. The RAD2 3' mRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination. Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light. The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nucleotide-long purine-rich tract. The sequence T G G A G G C A T T A A found at position -167 to -156 in the RAD2 gene is similar to a sequence present in the 5' flanking regions of the RAD7 and RAD10 genes.

Amino Acid Sequence↗
PubMed1975

Restriction and modification in B. subtilis. Nucleotide sequence recognised by restriction endonuclease R. Bsu R from strain R.

Restriction endonuclease R from Bacillus subtilis strain R cleaves nonmodified SPP 1 DNA in approximately 80, and lambda DNA in about 200 different sites. DNA digests with this endonuclease and with endonuclease Hae III from Haemophilus aegyptius show identical fragmentation patterns on gel electrophoresis, indicating that the two enzymes recognise the same nucleotide sequence. The polynucleotide kinase reaction was used in conjunction with two-dimensional ionophoretic nucleotide mapping methods to identify the 5'-nucleotide sequences at the sites of cleavage by the B. subtilis restriction endonuclease. The results show that the recognition sequence is (see article) where arrows indicate the points of strand scission. Each of the four possible nucleotides can occur in the positions flanking the recognition site.

Bacillus subtilis↗
PubMed1996

Mapping of branchpoint nucleotides in mutant pre-mRNAs expressed in plant cells.

Pre-mRNA encoding rubisco activase in the Arabidopsis thaliana mutant rca contains a GU to AU change at the 5' splice site of intron 3 and this mutation results in accumulation of splicing intermediates bearing an incompletely processed intron. It has been demonstrated that one of the intermediates contains intron 3 in the form of a lariat and the branchpoint nucleotide has been mapped to the A residue at position -32 forming part of the sequence UUGAU. Analysis of a similar GU to AU 5' splice site mutation, present in a synthetic pre-mRNA context expressed in transfected protoplasts of Nicotiana plumbaginifolia, also suggests formation of lariats with branching occurring at A-31. A small fraction (approximately 10%) of this mutant pre-mRNA also underwent the second step of splicing. In addition to the consensus AG, an AU dinucleotide was used as splicing acceptor.

Arabidopsis↗
PubMed2005

Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event.

Basal cell carcinoma is the most common human cancer with increasing incidence reported worldwide. Despite the aberrant signaling role of the Hedgehog pathway, little is known about the genetic mechanisms underlying basal cell carcinomas. Towards a better understanding of global genetic events, we have employed the Affymetrix Mapping 10K single nucleotide polymorphism (SNP) microarray technique for "fingerprinting" genomewide allelic imbalance in 14 basal cell carcinoma-blood pair samples. This rapid high-resolution SNP genotyping technique has revealed a somatic recombination event-uniparental disomy, leading to a loss of heterozygosity (LOH), as a key alternative genetic mechanism to allelic imbalances in basal cell carcinomas. A highly conserved LOH region at 9q21-q31 was found in 13 of 14 (93%) basal cell carcinomas. Further statistical and fluorescence in situ hybridization analyses confirmed that the 9q LOH was a result of uniparental disomy in 5 of 13 (38%) basal cell carcinomas. De novo mutations in the Patched 1 gene (PTCH) were found in 9 of 13 (69%) basal cell carcinomas with 9q LOH. A second important locus, containing LOH at 6q23-q27 was found in 5 of 14 (36%) basal cell carcinomas, suggesting that the presence of an additional putative tumor suppressor gene may be contributing to basal cell carcinoma development. This study shows that the rate of 9q LOH in basal cell carcinomas has been previously underestimated. Furthermore, we provide the first evidence that uniparental disomy due to somatic recombination constitutes one of the mechanisms of LOH in basal cell carcinoma tumorigenesis.

Aged↗