Analysis of human immunodeficiency virus type 1 integrase mutants.
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Base Sequence↗
Biomedical subjects
R A Gibbs
Publications and source records attributed to R A Gibbs.
Analysis of human immunodeficiency virus type 1 integrase mutants.
The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication.
Base Sequence↗
Detection and genetic analysis of beta-thalassemia mutations by competitive oligopriming.
A new approach for the detection of beta-thalassemia mutations has been applied, based on competitive oligonucleotide priming (COP) of in vitro DNA amplification at the mutation site. This method allows genotyping of the template DNA, through differential labeling of the allele-specific competitive oligoprimers and biotinylation of the common reverse primer. The system provides a basis for rapid, simple, and reliable detection of the numerous known beta-thalassemia mutations, revealing the precise nature of the mismatch in each case, and thereby facilitating the molecular genetic analysis of the disease.
Base Sequence↗
Sixty-nine kilobases of contiguous human genomic sequence containing the alpha-galactosidase A and Bruton's tyrosine kinase loci.
Several disease loci have been mapped to the Xq21.3-Xq22 region of the human X Chromosome (Chr) including X-linked agammaglobulinemia (XLA), Fabry disease, Alport syndrome, and Pelizaeus Merzbacher disease. Upon cloning of the XLA gene, Bruton's tyrosine kinase (btk), both Fabry disease and XLA were mapped within the same 50- to 70-kb interval. In order to investigate the genomic organization of the region surrounding btk and the Fabry disease gene, alpha-galactosidase A (gla), we constructed a 6-cosmid contig spanning the region from 5' of gla to 3' of btk. Two of these cosmids spanning most of the coding sequence and the upstream region of btk and gla, U237D10 and U230D1, were sequenced by a random shotgun strategy combined with automated sequencing, resulting in 69 kb of contiguous genomic sequence. Sequencing of U237D10 showed btk to be comprised of 19 exons spanning over 35 kb. Sequencing of U230D1 showed that the 3' end of gla is 9 kb from the 5' end of btk and also demonstrated the presence of two additional genes in the region immediately 5' to btk. The surprisingly high gene density is similar to that seen previously only in the human major histocompatibility locus.
Agammaglobulinaemia Tyrosine Kinase↗
Working on the assembly line.
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Base Sequence↗
Pressing ahead with human genome sequencing.
While waiting for revolutionary techniques to emerge, conventional approaches have been used to generate more than 40 million bases of DNA sequence from large scale projects involving different organisms. About eight megabases are from the human genome. Does this progress signal the time to move ahead for a massive human genomic sequencing effort, or should we bide our time, waiting for more technical developments? The answer is to move ahead now, choosing carefully those strategies that can be easily adapted to future methodologies.
Animals↗
Accurate determination of DNA in agarose gels using the novel algorithm GelScann(1.0).
GelScann(1.0) is a user-friendly program that accurately quantitates DNA from CCD imaged agarose gels. The algorithm automatically locates lanes, locates bands within a given lane, and quantitates the intensity of each band. GelScann (1.0) uses a statistical method to discriminate DNA from local background fluorescence. The sensitivity of the LaneFinder and BandFinder can be adjusted by the user interacting with GelScann (1.0)'s option box. We demonstrate that GelScann(1.0) can accurately determine DNA that GelScann(1.0) can accurately determine DNA concentrations for routine molecular biology applications and determine the relative intensities of PCR amplified fragments for genetic testing.
Algorithms↗
130 kb of DNA sequence reveals two new genes and a regional duplication distal to the human iduronate-2-sulfate sulfatase locus.
Deficiency of IDs activity results in Hunter Syndrome (mucopolysaccharidosis type II), a fatal X-linked recessive disorder. We report characterization of 28 cosmids around the IDS locus in Xq28. Four overlapping cosmids have been sequenced in their entirety generating a 130-kb contig. These studies show the fine structure of the IDS gene and identify an IDS pseudogene-like structure located 20 kb distal to the active gene. Two novel genes have also been identified in this sequence, and one of these genes is also locally duplicated. Both homologs are expressed, and a number of alternative transcript products have been characterized. The presence of a highly conserved pseudogene-like structure within a larger duplicated region close to the IDS gene has significant implications for the study of mutations at this locus.
Base Sequence↗
Complete sequence of a 38.4-kb human cosmid insert containing the polymorphic marker DXS455 from Xq28.
The complete DNA sequence of a cosmid mapping to human Xq28 (DXS455) has been determined using a shotgun approach (genbank accession number: L31948). The cosmid insert is 38.4 kb in length, and contains several repetitive sequences, including a highly repetitive region located in a fragment previously shown to contain a polymorphic VNTR locus, as well as short di- and tri-nucleotide repeats. In addition to the previously known VNTR locus, a CA-repeat and an unusual 800-bp repetitive region have been found to be polymorphic. The repeated sequence mapping to the site of the VNTR locus spans 480 bp in the cosmid. In addition to several AT-dinucleotide stretches, this repeat has four copies of a 25 bp repeat unit and flanking sequences similar to this 25-mer. No expressed sequences have so far been identified in this cosmid.
Bacteriophage M13↗
Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.
Base Sequence↗
Adaptor-based uracil DNA glycosylase cloning simplifies shotgun library construction for large-scale sequencing.
An improved strategy for the preparation of libraries for the random sequencing of DNA is reported. The protocol is a modification of a previous adaptor-based strategy, and utilizes long (11 base) overhangs, which eliminates the unreliable step of vector-insert ligation. The random inserts are prepared by adaptor ligation, while the M13 vector is prepared as described for uracil DNA glycosylase (UDG) cloning of polymerase chain reaction (PCR) products, using PCR with uracil-containing primers, followed by UDG treatment to produce overhangs. This method has been found to reliably yield large numbers of clones. There is no background due to religation of the vector, and all clones contain inserts. In addition, the method is simple and suitable for export to other investigators. Libraries were constructed from cosmids containing human DNA and from human cDNAs in order to characterize a strategy for shotgun sequencing of multiple shorter fragments.
Base Sequence↗
Analysis of HIV type 1 reverse transcriptase expression in a human cell line.
The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.
Base Sequence↗
Multiplex PCR: advantages, development, and applications.
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Animals↗
Fine structure of the human FMR1 gene.
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Base Sequence↗
Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.
The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.
Adult↗
Large-scale sequencing library production: an adaptor-based strategy.
A significant bottleneck in large-scale DNA sequencing projects is the creation of high-quality random libraries suitable for sequencing. We have utilized an adaptor-based strategy and developed a simple protocol to reliably generate single-insert random subclone M13 libraries yielding uniform coverage and > 10(3) clones per microgram of DNA. Of the resulting white plaques, 95% contained inserts, < 0.5% were due to adaptors with no insert, and none of the clones were found to contain extraneous DNA (0/600) or multiple inserts (0/300).
Base Sequence↗
Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.
We report here the development of multiplex in vitro DNA amplification and solid-phase direct exon sequencing for the analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. 18 representative HPRT-deficient mutants, derived either spontaneously, or after exposure to UV light or ionizing radiation, were analyzed. All 9 hprt exons were simultaneously amplified via the polymerase chain reaction (PCR) for rapid deletion detection. 5 mutants involve single- or multiple-exon deletions. Altered multiplex PCR patterns were detected in mutants Bsp-040, Bsp-065 and BGR-606. Subsequent direct sequence analysis reveals that Bsp-040 and Bsp-065 carry a 52-bp and a 13-bp intragenic DNA deletion in exon 3, respectively. BGR-606 contains a 223-bp insertion accompanied by a 10-bp deletion of intron sequence within exon 4 fragment. Other subtle DNA alterations identified by direct exon sequence analysis include single-base substitutions, small deletions and insertions, and RNA splicing mutations.
Animals↗
Fine structure of the human FMR1 gene.
The fragile X syndrome is due to a CGG triplet expansion in the first exon of FMR1, resulting in hypermethylation and extinction of gene expression. To further our understanding of the gene's involvement in the syndrome, we report the physical structure of this locus. A high resolution restriction map of the FRAX(A) locus has been prepared encompassing approximately 50 kb. Using exon-exon PCR and restriction analysis, the FMR1 gene has been determined to consist of 17 exons spanning 38 kb of Xq27.3. Each intron-exon boundary has been sequenced. In general, the splice donors and acceptors located in the 5' portion of the gene demonstrate greater adherence to consensus than those in the 3' end, providing a possible explanation for the finding of alternative splicing in FMR1. The elucidation of the exon composition of the FMR1 gene and its flanking region will enhance detection of coding sequence mutations possible in fragile X phenocopy individuals.
Alternative Splicing↗