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Phillip B Danielson

Publications and source records attributed to Phillip B Danielson.

8 recordsLinked to original sources
PubMed2005

Separating human DNA mixtures using denaturing high-performance liquid chromatography.

DNA mixtures represent challenging samples that are rarely amenable to direct DNA sequence analysis and many of the strategies available to separate mixtures are both labor and time intensive. Denaturing high-performance liquid chromatography is an accurate and rapid approach for the detection and scoring of mutations. It can also be used to separate DNA mixtures. The technique relies on the chromatographic separation of crosshybridization products to isolate the individual components of a mixture. By eliminating secondary amplification and excessive manipulation prior to sequencing, denaturing high-performance liquid chromatography can streamline the analysis of conditions ranging from somatic mosaicism, microchimerism and mitochondrial heteroplasmy to evidentiary material containing mixtures of DNA encountered in forensic investigations.

Chimerism↗
PubMed2003

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

Amino Acid Sequence↗
PubMed2003

Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC.

A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e., orders Acipenseriformes and Semionotiformes), certain extant lobe-finned fish (Australian lungfish and African lungfish), and the tetrapods there are three melanocortin regions in POMC: ACTH/alphaMSH, beta-MSH, and gamma-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the gamma-MSH sequence is absent from the POMC genes of euteleosts like the carp, tilapia, chum salmon, sockeye salmon, and rainbow trout. The objective of this study was to determine whether the gamma-MSH sequence still may be present in the POMC gene of a more basal lineage of the teleosts such as a representative from subdivision Elopomorpha. To this end, a POMC cDNA was cloned and sequenced from the pituitary of the American eel, Anguilla rostrata (order Anguilliformes, family Anguillidae). The open reading frame of the eel POMC cDNA was 648 nucleotides in length and encoded 216 amino acids. As predicted, eel POMC contained the deduced amino acid sequences for beta-endorphin, ACTH/alpha-MSH, and beta-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a gamma-MSH sequence and a large portion of the joining peptide region. In this regard, the eel POMC gene thus displays features very similar to the POMC genes that have been sequenced from euteleosts. Although it is conceivable that the gamma-MSH sequence may be present in representatives from the other basal extant lineages of teleosts (i.e., subdivisions Osteoglossomorpha or Clupeomorpha), it is also possible that the deletion that resulted in the loss of the gamma-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the gamma-MSH sequence should be absent in all extant teleosts.

Amino Acid Sequence↗
PubMed2003

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

Adrenocorticotropic Hormone↗
PubMed2003

Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.

AIM: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA). METHODS: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method. RESULTS: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches. CONCLUSIONS: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution.

Blood Stains↗
PubMed2003

Clinical applications of denaturing high-performance liquid chromatography-based genotyping.

AIM: To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system. METHODS: Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis. RESULTS: Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans. CONCLUSION: DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Analyzing the evolution of the opioid/orphanin gene family.

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell. To understand the functional capabilities of the secretory cell, the molecular genetics approaches must be combined with procedures that actually characterize the end-products generated by the secretory cell. Recent advances in two-dimensional gel electrophoresis and mass spectrometry now make it possible to analyze neuropeptides from a relatively small amount of tissue. These procedures can reveal novel end-products, tissue-specific endoproteolytic cleavage events, and developmental shifts in post-translational processing schemes. A gene family that illustrates all of these processes and the advantages of combining genomics with proteomics is the opioid/orphanin gene family.

Amino Acid Sequence↗