Sequencing with the large fragment of DNA polymerase I from<i>Bacillus stearothermophilus</i>

McClary, John; Ye, S Y; Hong, Guofan; Witney, Frank

doi:10.3109/10425179109020768

Cited by 34 publications

(31 citation statements)

References 6 publications

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“…Plasmids-All hGALT mutations were recreated by site-directed mutagenesis of the otherwise wild-type sequence, as described previously (15). The primers used to generate alleles R67C, L139P, P183T, R201H, R231H, R259W, K285N, E291K, Y323D, and T350A were hGR67CF (5Ј-GAAGACAGTGCCCTGCCATGACCCTCTC-3Ј), hGL139PF (5Ј-GGATGTAACGCCGCCACTCATGTCG-3Ј), hGP183TF (5Ј-GCTGTTCTAACACCCACCCCCACT-3Ј), hGR201HF (5Ј-GATATT-GCCCAGCATGAGGAGCGA-3Ј), hGR231H (5Ј-TCAGGAAGGAACAT-CTGGTCCTAAC-3Ј), hGR259WF (5Ј-GCTGCCCCGTTGGCATGTGC-GGCGG-3Ј), hGK285NF (5Ј-GCTCTTGACCAATTATGACAACCTC-3Ј), hGE291KF (5Ј-GACAACCTCTTTAAGACGTCCTTTCC-3Ј), hGY323DF (5Ј-CACGCTCATTACGACCCTCCGCTC-3Ј), hGT350AF (5Ј-GAGGGA-CCTCGCCCCTGAGCAGGCT-3Ј), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…At 31 h after inoculation, a 6-ml sample was removed from each culture (zero time point), and galactose was added to the remaining volume of one culture from each pair to a final concentration of 0.05%. At 7,15, and 63 h after the addition of galactose, 6-ml samples from each culture were harvested, pelleted, and frozen. Finally, cell pellets were lysed as described above, except that protease inhibitors were not included in the lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

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Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Riehman

Crews

Fridovich‐Keil³

2001

Journal of Biological Chemistry

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Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1, 2). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2, 3).Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30 -50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2 and 3), the biochemical mechanism of pathophysiology in galactosemia remains unknown.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Riehman

Crews

Fridovich‐Keil³

2001

Journal of Biological Chemistry

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“…For yeast expression of the unfused AtKAP␣, the GAL4 activation domain was removed from pGADAtKAP␣ by oligonucleotide-directed mutagenesis (19), producing the pAt-KAP␣ construct. For expression of the SRP1 from Saccharomyces cerevisiae, PCR was used to introduce BamHI restriction sites at both 5Ј and 3Ј ends of the SRP1 ORF (20).…”

Section: Methodsmentioning

confidence: 99%

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein

Ballas

Citovsky

1997

Proc. Natl. Acad. Sci. U.S.A.

173

147

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T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a singlestranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5 end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAP␣, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAP␣ interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of At-KAP␣ was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin ␣ family. Indeed, AtKAP␣ efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAP␣ specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

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“…Site-directed Mutagenesis-Site-directed mutagenesis of pCMVhGR according to the refined method of Kunkel (25,26) was used to construct the mutants. The mutant plasmids were transformed into Escherichia coli by electroporation, minipreps of DNA (Wizard miniprep, Promega, Madison, WI) were made, and dideoxy sequencing was performed to confirm the mutations.…”

Section: Materials-[mentioning

confidence: 99%

Functional Probing of the Human Glucocorticoid Receptor Steroid-interacting Surface by Site-directed Mutagenesis

Lind

Greenidge

Gillner

et al. 2000

Journal of Biological Chemistry

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To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17␣-OH group. This effect was alleviated by the presence of a 16␣-CH 3 group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid. Met-560 and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point. The glucocorticoid receptor (GR)1 belongs to the superfamily of hormone-dependent nuclear receptors and consists of three structural and functional main domains: the N-terminal domain, which harbors the major transactivating function (AF-1); the central domain, which binds to DNA in glucocorticoid regulated genes; and the C-terminal domain, which binds the ligand (1-4).The ligand-binding domain (LBD) comprises approximately 250 amino acids and is in its unliganded state associated with a complex containing heat shock proteins and immunophilins (5). Upon ligand binding this complex dissociates and a cascade of events are triggered leading to induction or repression of target genes. Within the ligand-binding domain there are also a hormone-dependent nuclear localization signal (6) and hormone-dependent transactivation functions (AF-2) (7-10).The crystal structure of the GR LBD is not yet available, but the crystal structures of the LBDs of other members of the nuclear receptor superfamily including the peroxisome proliferator activated receptor, retinoic acid receptor, retinoid X receptor, thyroid hormone receptor, progesterone receptor (PR), estrogen receptor ␣ (ER␣), and estrogen receptor ␤ (ER␤) have been solved (11-17). Their structures contain 12 ␣-helices that are folded in a very similar way into a three-layered antiparallel ␣-helical sandwich that creates a hydrophobic pocket for the ligand. Upon ligand binding a conformational change occurs, mainly involving helix 12, which folds up against the protein body and creates a lid for the ligand binding pocket. This also leads to formation of the AF-2 interface, which has been shown to interact with transcriptional coactivators (18 -21).The mechanisms that determine the binding affi...

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Sequencing with the large fragment of DNA polymerase I fromBacillus stearothermophilus

Cited by 34 publications

References 6 publications

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein

Functional Probing of the Human Glucocorticoid Receptor Steroid-interacting Surface by Site-directed Mutagenesis

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