Reference DNA Sequence Research Articles

Sequencing the entire genomes of free-living organisms: the foundation of pharmacology in the new millennium.

The power and effectiveness of clinical pharmacology are about to be transformed with a speed that earlier in this decade could not have been foreseen even by the most astute visionaries. In the very near future, we will have at our disposal the reference DNA sequence for the entire human genome, estimated to contain approximately 3.5 billion bp. At the same time, the science of whole genome sequencing is fostering the computational science of bioinformatics needed to develop practical applications for pharmacology and toxicology. Indeed, it is likely that pharmacology, toxicology, bioinformatics, and genomics will merge into a new branch of medical science for studying and developing pharmaceuticals from molecule to bedside.

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype.

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.

Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides.

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey.

Depletion of yeast RNase III blocks correct U2 3' end formation and results in polyadenylated but functional U2 snRNA.

Yeast U2 snRNA is transcribed by RNA polymerase II to generate a single non-polyadenylated transcript. A temperature-sensitive yeast strain carrying a disruption in RNT1, the gene encoding a homolog of RNase III, produces 3'-extended U2 that is polyadenylated. The U2 3'-flanking region contains a putative stem-loop that is recognized and cleaved at two sites by recombinant GST-Rnt1 protein in vitro. Removal of sequences comprising the stem-loop structure blocks cleavage in vitro and mimics the effects of Rnt1 depletion in vivo. Strains carrying a U2 gene lacking the Rnt1 cleavage site produce only polyadenylated U2 snRNA, and yet are not impaired in growth or splicing. The results suggest that eukaryotic RNase III may be a general factor in snRNA processing, and demonstrate that polyadenylation is not incompatible with snRNA function in yeast.

DNA Molecules as Standard Reference Materials I: Development of DNA Identification Sequences and Human Mitochondrial DNA Reference Sequences.

This paper describes the design, construction and characterization of two DNA molecules that function as prototype Standard Reference Materials for use in the determination of DNA sequences. The first prototype reference DNA molecule was constructed to function as a true reference DNA sequence encoding the human mitochondrial hypervariable region 1. The second DNA molecule was designed to be a molecular DNA “tag” for use in the identification of DNA Standard Reference Materials. DNA molecules were chemically and enzymatically synthesized, cloned in molecular cloning vectors and sequenced using manual and automated fluorescent DNA sequencing methods. The sequence of the cloned human mitochondrial HV1 DNA shows six base pair differences from the original published DNA sequence. The molecular DNA tag sequence uses the universal genetic code with one letter amino acid abbreviations to spell the words “NIST DNA STANDARD REFERENCE MATERIAL.” The need and utility of an expanded molecular code to uniquely identify DNA molecules is discussed. The use of these DNA molecules in the development of DNA Standard Reference Materials is also described.

Durchlässigkeit der Siebröhren für Druckströmungen.

Mollisia is a taxonomically neglected discomycete genus (Helotiales, Leotiomycetes) of commonly encountered saprotrophs on decaying plant tissues throughout temperate regions. The combination of indistinct morphological characters, more than 700 names in the literature, and lack of reference DNA sequences presents a major challenge when working with Mollisia. Unidentified endophytes, including strains that produced antifungal or antiinsectan secondary metabolites, were isolated from conifer needles in New Brunswick and placed with uncertainty in Phialocephala and Mollisia, necessitating a more comprehensive treatment of these genera. In this study, morphology and multigene phylogenetic analyses were used to explore the taxonomy of Mollisiaceae, including Mollisia, Phialocephala, and related genera, using new field collections, herbarium specimens, and accessioned cultures and sequences. The phylogeny of Mollisiaceae was reconstructed and compared using the nuc internal transcribed spacer rDNA (ITS) barcode and partial sequences of the 28S nuc rDNA (LSU) gene, largest subunit of RNA polymerase II (RPB1), DNA topoisomerase I (TOP1), and the hypothetical protein Lipin/Ned1/Smp2 (LNS2). The results show that endophytism is common throughout the Mollisiaceae lineage in a diverse range of hosts but is infrequently attributed to Mollisia because of a paucity of reference sequences. Generic boundaries within Mollisiaceae are poorly resolved and based on phylogenetic evidence the family included species placed in Acephala, Acidomelania, Barrenia, Bispora, Cheirospora, Cystodendron, Fuscosclera, Hysteronaevia, Loramyces, Mollisia, Neopyrenopeziza, Obtectodiscus, Ombrophila, Patellariopsis, Phialocephala, Pulvinata, Tapesia (=Mollisia), and Trimmatostroma. Taxonomic novelties included the description of five novel Mollisia species and five novel Phialocephala species and the synonymy of Fuscosclera with Phialocephala, Acidomelania with Mollisia, and Loramycetaceae with Mollisiaceae.