Applications of NMR spectroscopy to studies of reactive intermediates and their interactions with nucleic acids.
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Base Sequence↗
Biomedical subjects
C M Harris
Publications and source records attributed to C M Harris.
The efficiency of translesion synthesis past single styrene oxide DNA adducts in vitro is polymerase-specific.
In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced at four different base positions within a sequence containing codons 60-62 from the human N-ras gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase alpha, and polymerase beta. Replication of the damaged templates was analyzed under conditions defining single and/or multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed at the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3' to the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or "fingerprints", that were unique to each polymerase.
Animals↗
Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine.
Improved methodology has been developed for preparation of oligodeoxynucleotides bearing adducts on the N2 position of guanine in which the adduction reaction is carried out in homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix. The methodology utilizes a new synthon, 2-fluoro-O6-(trimethylsilylethyl)-2'-deoxyinosine (3). Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O6 protection is eliminated under very mild conditions following displacement of the 2-fluoro group by amine nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed by removal of the remaining protective groups with NH4OH (60 degrees C, 8 h) and then 0.1% acetic acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting groups on the exocyclic amino groups.
Base Sequence↗
Comparison of the efficiency of synthesis past single bulky DNA adducts in vivo and in vitro by the polymerase III holoenzyme.
Previous studies from our laboratory revealed that site-specific and stereospecific styrene oxide (SO) lesions in M13 DNA were readily bypassed when transfected into Escherichia coli cells, but these same lesions blocked the progress of several purified polymerases in vitro when situated in oligodeoxynucleotide templates (Latham, G. J., et al. (1993) J. Biol. Chem. 268, 23427-23434; Latham, G. J., et al. (1995) Chem. Res. Toxicol. 8, 422-430). To resolve this apparent discrepancy, we constructed single-stranded M13 genomes containing single SO adducts and compared their replication efficiencies in E. coli cells to the extent of bypass synthesis in vitro using three different complexes of the purified E. coli polymerase III (Pol III) holoenzyme. The transformation efficiencies of the SO-adducted M13 templates were comparable to those of the nonadducted controls, indicating facile bypass in E. coli. When the identical adducted M13 vectors were replicated in vitro with the reconstituted complexes of the Pol III holoenzyme, the results were consistent with the in vivo data: Synthesis past two of the three SO adducts in M13 was unhindered relative to synthesis on the unadducted M13 control template. Since our previous in vitro assays indicated that SO adducts in 33-mer templates largely blocked polymerases other than Pol III, we repeated these studies using reconstituted Pol III. Significantly, Pol III replication was poorly processive and strongly terminated by SO lesions in 33-mer templates. This result was in stark contrast to the efficient bypass in vitro of the same adducts in M13 DNA. In fact, Pol III-mediated bypass was enhanced to > 75-fold on adducted circular M13 templates as compared to adducted linear oligodeoxynucleotides. The implications of the effects of polymerase processivity and template-primer structure upon lesion bypass are discussed.
Bacteriophage M13↗
Medical stereotypes.
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Attitude of Health Personnel↗
Electrophysiological and eye-movement abnormalities in children with the Bardet-Biedl syndrome.
We investigated 17 patients with clinical features of Bardet-Biedl syndrome by electroretinogram (ERG), visual-evoked potentials (VEP), and electro-oculographic (EOG) eye movement assessment. The ERGs were grossly abnormal in 16 cases. Pattern VEPs were generally well preserved, but showed a tendency to increase in latency and decrease in amplitude with age. These results confirm other reports that the retinopathy appears to be a progressive rod-cone dystrophy initially affecting mainly extramacular areas but involving the macula at later stages. Seven of 11 patients showed abnormalities of either optokinetic nystagmus, vestibulo-ocular reflex, or both. These eye-movement abnormalities have not been reported previously, and are further evidence of central nervous system involvement in this syndrome.
Adolescent↗
Knee fusion with intramedullary rods for failed total knee arthroplasty.
Knee fusion was obtained after failed total knee arthroplasty with the aid of internal fixation by use of intramedullary rods. The method has been used in eight cases, including difficult situations characterized by morbid obesity, segmental fractures, and loss of bone stock. In these cases, stability would have been difficult to obtain with conventional methods of external fixation, compression, or casting. Stability was achieved quickly with an intramedullary rod in eight cases; fusion was obtained in seven.
Aged↗
Todd and teaching from the point of view of general practice.
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Education, Medical↗
Gq-protein alpha subunit expression and distribution in pregnant rat myometrial tissues.
OBJECTIVE: Multiple G-protein isoforms play an integral role in signal transduction; the Gq subtype of G-protein alpha subunits is involved in the activation of the phosphatidylinositol signaling pathway. The studies described herein evaluate the expression of Gq, along with Gs and Gi, in pregnant and nonpregnant rat myometrial tissues. METHODS: Myometrium and other tissues were obtained from nonpregnant and timed-pregnant Sprague-Dawley rats. Western blot studies were performed using polyclonal G-protein isoform-specific antibodies. Immunohistochemical studies were performed using the same antibodies with specimens of myometrium, intestine, and skeletal muscle. RESULTS: The Western blot studies confirmed differential expression of all types of G-protein alpha subunit subtypes in rat myometrial tissues. In pregnant rat myometrium, the expression of Gq and Gs was sustained through day 22, whereas, Gi expression decreased on day 20 and remained low through the remainder of gestation. The immunohistochemical studies revealed significant staining for Gq and Gs in the myometrial layers of the pregnant and nonpregnant rat uterus; in contrast, immunostaining for Gi was minimal in nonpregnant myometrium, and even lower in myometrium from pregnant uteri. CONCLUSIONS: These studies have confirmed expression of the Gq, Gi, and Gs alpha subunits in rat myometrial tissue. Immunohistochemistry confirmed that Gq was expressed at high levels in the myometrial layer of the pregnant and nonpregnant uterus. These observations support the hypothesis that Gq expression is critically important for the transduction of hormone signals, such as those responsible for the generation of phasic myometrial contractions.
Animals↗