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Biomedical subjects

R A Gibbs

Publications and source records attributed to R A Gibbs.

At least 19 recordsLinked to original sources
PubMed1999

An allylic/acyclic adenosine nucleoside triphosphate for termination of DNA synthesis by DNA template-dependent polymerases.

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2',3'-dideoxyadenosine-5'-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.

Adenosine Triphosphate↗
PubMed1999

Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors.

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).

Alkyl and Aryl Transferases↗
PubMed1998

The genomic organization of Isopeptidase T-3 (ISOT-3), a new member of the ubiquitin specific protease family (UBP).

A novel Isopeptidase T gene (ISOT-3) has been identified on human mosome 3q26.2--q26.3. gene shows 67.3% nucleotide identity and 54.8% amino acid identity to n Isopeptidase (ISOT-1). Northern blot analysis has shown that ISOT-3 is highly essed in ovary and testes, low-level expression in six other tissues tested. In contrast, ISOT-1 is essed at high levels in brain, and there is no detectable expression in ovary. The exonic nization of these two genes highly conserved with only one variant intron position. Intron 15 in -3 is absent in ISOT-1, there is an alternate splice site at the same location. Although the --intron structure has been erved between the two genes, ISOT-3 has significantly larger intronic ons, and the overall of this gene is at least 90 kb compared to 15 kb for ISOT-1. These data suggest that both ISOT-1 and ISOT-3 have descended from a common ancestor. In addition, the low overall sequence identity and different expression patterns may reflect differences in substrate specificity.

Amino Acid Sequence↗
PubMed1998

DNA deletion confined to the iduronate-2-sulfatase promoter abolishes IDS gene expression.

Deficiency of the enzyme iduronate-2-sulfatase (IDS) results in Hunter syndrome, an X-linked recessive lysosomal storage disorder. In this study, analysis of a patient with features of moderate to severe Hunter syndrome identified a 178-bp deletion upstream of IDS exon 1 spanning a predicted promoter element. Sequencing of all nine IDS exons from this patient failed to identify any additional mutations within the coding regions or in intron-exon boundaries. The 178-bp deletion is flanked by two 13-bp direct repeats and potential DNA topoisomerase II recognition sites. These findings point toward nonhomologous recombination as a possible mechanism for this mutation. Expression studies on this patient do not detect any IDS transcripts, indicating that the deletion spans sequences essential for IDS expression. Complete lack of expression of IDS is consistent with the moderate to severe phenotype observed in this patient.

Base Sequence↗
PubMed1998

Analysis of the quality and utility of random shotgun sequencing at low redundancies.

The currently favored approach for sequencing the human genome involves selecting representative large-insert clones (100-200 kb), randomly shearing this DNA to construct shotgun libraries, and then sequencing many different isolates from the library. This method, entitled directed random shotgun sequencing, requires highly redundant sequencing to obtain a complete and accurate finished consensus sequence. Recently it has been suggested that a rapidly generated lower redundancy sequence might be of use to the scientific community. Low-redundancy sequencing has been examined previously using simulated data sets. Here we utilize trace data from a number of projects submitted to GenBank to perform reconstruction experiments that mimic low-redundancy sequencing. These low-redundancy sequences have been examined for the completeness and quality of the consensus product, information content, and usefulness for interspecies comparisons. The data presented here suggest three different sequencing strategies, each with different utilities. (1) Nearly complete sequence data can be obtained by sequencing a random shotgun library at sixfold redundancy. This may therefore represent a good point to switch from a random to directed approach. (2) Sequencing can be performed with as little as twofold redundancy to find most of the information about exons, EST hits, and putative exon similarity matches. (3) To obtain contiguity of coding regions, sequencing at three- to fourfold redundancy would be appropriate. From these results, we suggest that a useful intermediate product for genome sequencing might be obtained by three- to fourfold redundancy. Such a product would allow a large amount of biologically useful data to be extracted while postponing the majority of work involved in producing a high quality consensus sequence.

Animals↗
PubMed1998

Reassessment of biochemically determined Hunter syndrome carrier status by DNA testing.

Deficiency of iduronate-2-sulphatase (IDS) results in the X linked recessive lysosomal storage disorder Hunter syndrome. Determination of carrier status in families affected by this disorder has been performed using a variety of enzymatic tests. None of these tests has proved to be 100% effective at identifying carriers. The aim of this study was to perform carrier testing in a family affected by the disorder, where testing was complicated by the fact that no surviving affected subjects were available for study. Direct dye primer sequencing of PCR products was used to identify mixed bases in an obligate carrier. Two mixed bases were observed within exon VIII. The first base change (T-->A) at nucleotide position 1150 results in a missense mutation (H342Q), while the second base change (G-->T) at nucleotide position 1151 results in a nonsense mutation (G343X). Four additional female family members were screened for the same mutation. Using this approach it is possible to provide unambiguous information about a subject's carrier status and, unlike biochemical testing, this approach will be equally effective when applied to families with the mild form of this disorder.

DNA↗
PubMed1998

Elimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up.

Here, we describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the base addition sequencing scheme (BASS).

Base Sequence↗
PubMed1998

Comparative sequence analysis of a gene-rich cluster at human chromosome 12p13 and its syntenic region in mouse chromosome 6.

The Human Genome Project has created a formidable challenge: the extraction of biological information from extensive amounts of raw sequence. With the increasing availability of genomic sequence from other species, one approach to extracting coding and regulatory element information is through cross-species sequence comparison. To assess the strengths and weaknesses of this methodology for large-scale sequence analysis, 227 kb of mouse sequence syntenic to a gene-rich cluster on human chromosome 12p13 was obtained. Primarily through percent identity plots (PIPs) of SIM comparative sequence alignments, the sequence of coding regions, putative alternative exons, conserved noncoding regions, and correlation in repetitive element insertions were easily determined. The analysis demonstrated that the number, order, and orientation of all 17 genes are conserved between the two species, whereas two human pseudogenes are absent in mouse. In addition, apart from MIRs, no direct correlation of distribution or position of the majority of repetitive elements between the two species is seen. Finally, in examining the synonymous and nonsynonymous substitution rates in the conserved genes, a large variation in nonsynonymous rates is observed indicating that the genes in this region are diverging at different rates. This study indicates the utility and strength of large-scale cross-species sequence comparisons in the extraction of biological information from raw sequence, especially when combined with other computational tools such as GRAIL and BLAST.

Amino Acid Sequence↗
PubMed1998

Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.

Direct DNA sequencing of human immunodeficiency virus type 1 (HIV-1) pol and env gene regions was characterized for accuracy and precision. Restricted maximum likelihood (REML) analysis of molecular clone reconstruction experiments using a primer-walking strategy showed that the BODIPY and BODIPY energy-transfer (BET) dye primers sets were significantly more accurate in quantitating heterogenous base mixtures than the fluorescein/rhodamine dye primers. Of the three sets examined, the BET dye primers were the most accurate. The improved accuracy correlated with the reduced emission band-widths of BODIPY and BET dye primers and the more uniform signal intensities of BET dye primers. However, comparing % coefficients of variation (CV) for the three dye primer sets, revealed that BODIPY dye primers gave better precision than both BET and fluorescein/rhodamine dye primer sets. Several sequence-dependent motifs were identified that showed specific nucleotide-biased incorporation and were determined to be the major variable component of the total %CV. Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations.

Boron Compounds↗
PubMed1997

Test of the potential of a dATP surrogate for sequencing via MALDI-MS.

1-(2'-Deoxy-beta-d-ribofuranosyl)-3-nitropyrrole phosphate was incorporated into a DNA decamer and analyzed via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The extent and composition of the various fragment peaks were compared with those in the MALDI-MS spectrum of dT4AT5. The nitropyrrole-containing oligomer proved to be more robust. Two different DNA template assays were then used to attempt to identify DNA replicating enzymes that would incorporate the corresponding triphosphate, i.e. 1-(2'-deoxy-beta-d-ribofuranosyl)-3-nitropyrrole triphosphate (dXTP). It was shown that dXTP was not incorporated by some enzymes and it inhibited others. However, DNA polymerase I Klenow fragment and avian myeloblastosis virus reverse transcriptase incorporated dXTP in place of dATP and then replicated the template overhang in the usual way. The potential of dXTP as a surrogate for dATP in DNA sequencing with MALDI-MS analysis is discussed.

DNA Polymerase I↗
PubMed1997

Novel modifications to the farnesyl moiety of the a-factor lipopeptide pheromone from Saccharomyces cerevisiae: a role for isoprene modifications in ligand presentation.

The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone [YIIKGVFWDPAC(farnesyl)-OCH3] in which posttranslational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity. Utilizing novel synthetic techniques and a well-characterized array of biological assays, we prepared original modifications to the farnesyl moiety of the pheromone in order to assess the importance of this part of the lipopeptide for biological activity. Specifically, the 3-methyl group was replaced to create analogs containing the ethyl, vinyl, tert-butyl, and phenyl moieties at the 3-position of the farnesyl chain. Subsequent biological analyses demonstrated that all of these modifications render an active pheromone, with the vinyl and ethyl analogs exhibiting higher activity than the native a-factor. However, the level of activity varied with the modification; the bulkier and more hydrophobic groups (tert-butyl and phenyl) exhibited lower biological activity than the smaller moieties (ethyl and vinyl). Furthermore, two analogs with phenyl substitutions that differ only in the presumed isomerization of the allylic double bond show up to an 8-fold difference in bioactivity. It has previously been surmised that the role of isoprenoid additions is solely to target the attached polypeptides to membranes by increasing their hydrophobicity. However, these studies demonstrate that even modest structural changes to the isoprenoid can significantly affect biological activity. These results are clearly inconsistent with a simple hydrophobic role for the isoprenoid and instead illustrate that it plays an active role in mediating optimal a-factor/receptor interaction.

Butadienes↗
PubMed1997

Design and synthesis of chiral and racemic phosphonate-based haptens for the induction of aldolase catalytic antibodies.

A novel strategy for the generation of aldolase catalytic antibodies, based on the use of antibody-catalyzed enol ester hydrolysis as a 'trigger' to generate a reactive enolate intermediate, is described. A model system to test this strategy was developed and substrate 8 was synthesized. However, the targeted bifunctional haptens 11 and 33 were synthetically inaccessible, and therefore the alternative phosphonate hapten 39 was prepared. The key step in the synthesis of 39 was the direct generation of an unprotected phosphonate precursor via coupling of the secondary alcohol 37 with CH3P(O)Cl2. The chiral counterpart of hapten 39 was also synthesized from alcohol 46, prepared by Corey's asymmetric reduction method. One polyclonal antibody preparation generated from 39 appeared to catalyze the hydrolysis of the secondary acetate 49, but not the desired aldol cyclization of 8. Possible rationales for this finding are discussed.

Antibodies↗
PubMed1997

Molecular and phenotypic variation in patients with severe Hunter syndrome.

Severe Hunter syndrome is a fatal X-linked lysosomal storage disorder caused by iduronate-2-sulphatase (IDS) deficiency. Patients with complete deletion of the IDS locus often have atypical phenotypes including ptosis, obstructive sleep apnoea, and the occurrence of seizures. We have used genomic DNA sequencing to identify several new genes in the IDS region. DNA deletion patients with atypical symptoms have been analysed to determine whether these atypical symptoms could be due to involvement of these other loci. The occurrence of seizures in two individuals correlated with a deletion extending proximal of IDS, up to and including part of the FMR2 locus. Other (non-seizure) symptoms were associated with distal deletions. In addition, a group of patients with no variant symptoms, and a characteristic rearrangement involving a recombination between the IDS gene and an adjacent IDS pseudogene (IDS psi), showed normal expression of loci distal to IDS. Together, these results identify FMR2 as a candidate gene for seizures, when mutated along with IDS.

Chromosome Mapping↗
PubMed1997

The human COX10 gene is disrupted during homologous recombination between the 24 kb proximal and distal CMT1A-REPs.

The CMT1A-REPs are two large directly repeating DNA sequences located on chromosome 17p11.2-p12 flanking the region duplicated in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) and deleted in patients with hereditary neuropathy with liability to pressure palsies (HNPP). We have sequenced two cosmids, c74F4 and c15H12, which contain the entire proximal and distal CMT1A-REPs and determined that these repeats are approximately 99% identical across a 24,011 bp region. In addition, both contain an exon of the human heme A:farnesyltransferase gene (COX10). Hybridization studies revealed that COX10 spans the distal CMT1A-REP, while the proximal CMT1A-REP contains an isolated COX10 'pseudo-exon'. There is also a COX10 hybridization signal on chromosome 10 which appears to represent a processed pseudogene. We propose that the distal CMT1A-REP represents the progenitor copy of COX10 exon VI which was duplicated with surrounding intronic sequences during mammalian genome evolution and that the HNPP deletion results in a COX10 null allele.

Alkyl and Aryl Transferases↗
PubMed1997

Large-scale sequencing in human chromosome 12p13: experimental and computational gene structure determination.

The detailed genomic organization of a gene-dense region at human chromosome 12p13, spanning 223 kb of contiguous sequence, was determined. This region is composed of 20 genes and several other expressed sequences. Experimental tools including RT-PCR and cDNA sequencing, combined with gene prediction programs, were utilized in the analysis of the sequence. Various computer software programs were employed for sequence similarity searches and functional predictions. The high number of genes with diverse functions and complex transcriptional patterns make this region ideal for addressing challenges of gene discovery and genomic characterization amenable to large-scale sequence analysis.

Alternative Splicing↗
PubMed1997

Large-scale comparative sequence analysis of the human and murine Bruton's tyrosine kinase loci reveals conserved regulatory domains.

Large-scale genomic DNA sequencing of orthologous and paralogous loci in different species should contribute to a basic understanding of the evolution of both the protein-coding regions and noncoding regulatory elements. We compared 93 kb of human sequence to 89 kb of mouse sequence in the Bruton's tyrosine kinase (BTK) region. In addition to showing the conservation of both position and orientation of the five functionally unrelated genes in the region (BTK, alpha-D-galactosidase A, L44L, FTP-3, and FCI-12), the comparison revealed conservation of clusters of noncoding sequence flanking the first exon of each gene. Furthermore, in the sequence comparison at the BTK locus, the conservation of clusters of noncoding sequence extends throughout the locus; the noncoding sequence is more highly conserved in the BTK locus in comparison to the flanking loci. This suggests a correlation with the complex developmental regulation of expression of btk. To determine whether a highly conserved 3.5-kb segment flanking the first exon of BTK contains transcriptional regulatory signals, we tested various portions of the segment for promoter and expression activity in several appropriate cell lines. The results demonstrate the contribution of the conserved region flanking the first exon to the cell lineage-specific expression pattern of btk. These data show the usefulness of large scale sequence comparisons to focus investigation on regions of noncoding sequence that play essential roles in complex gene regulation.

Agammaglobulinaemia Tyrosine Kinase↗