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P H Patel

Publications and source records attributed to P H Patel.

At least 19 recordsLinked to original sources
PubMed2001

Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection.

Accurate transmission of DNA material from one generation to the next is crucial for prolonged cell survival. Following the discovery of DNA polymerse I in Escherichia coli, the DNA polymerase I class of enzymes has served as the prototype for studies on structural and biochemical mechanisms of DNA replication. Recently, a series of genomic, mutagenesis and structural investigations have provided key insights into how Pol I class of enzymes function and evolve. X-ray crystal structures of at least three Pol I class of enzymes have been solved in the presence of DNA and dNTP, thus allowing a detailed description of a productive replication complex. Rapid-quench stop-flow studies have helped define individual steps during nucleotide incorporation and conformational changes that are rate limiting during catalysis. Studies in our laboratory have generated large libraries of active mutant enzymes (8000) containing a variety of substitutions within the active site, some of which exhibit altered biochemical properties. Extensive genomic information of Pol I has recently become available, as over 50 polA genes from different prokaryotic species have been sequenced. In light of these advancements, we review here the structure-function relationships of Pol I, and we highlight those interactions that are responsible for the high fidelity of DNA synthesis. We present a mechanism for "flipping" of the complementary template base to enhance interactions with the incoming nucleotide substrate during DNA synthesis.

Amino Acid Sequence↗
PubMed2001

The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable.

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.

Amino Acid Motifs↗
PubMed2000

Multiple amino acid substitutions allow DNA polymerases to synthesize RNA.

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10(3)-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.

Amino Acid Substitution↗
PubMed2000

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity.

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.

Amino Acid Motifs↗
PubMed2000

DNA polymerase active site is highly mutable: evolutionary consequences.

DNA polymerases contain active sites that are structurally superimposable and highly conserved in sequence. To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquaticus DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8, 000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity. A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.

Amino Acid Sequence↗
PubMed1999

Evaluation of an unused 1952 Ridley intraocular lens.

PURPOSE: To evaluate an unused 1952 historic Ridley intraocular lens (IOL) brought to Bombay, India, in 1952 from an Oxford Ophthalmologic Conference in England and given to 1 of the authors during his residency. SETTING: Alcon Laboratories, Fort Worth, Texas, USA. METHODS: The Ridley IOL was evaluated at Alcon Laboratories, Inc., using the established procedures of its Intraocular R&D Laboratories. Various optical and physical aspects of the Ridley lens were evaluated including (1) dimensions, (2) weight, (3) power, (4) resolution efficiency and modulation transfer function (MTF), (5) surface sphericity by interferometry, (6) ultraviolet (UV)-visible transmission characteristic, (7) attenuated total reflectance (ATR)-Fourier transform infrared reflectance spectrum, and (8) cosmetics by visual inspection using light microscopy. RESULTS: This 8.5 mm diameter, 2.4 mm thick, 23 diopter biconvex IOL weighed 108 mg. The ATR spectrum, UV-visible transmission, and refractive index confirmed its poly-(methyl methacrylate) material. The 0.56 MTF value at 100 line pairs/mm, per the International Standards Organization--IOL Optics Standard, and 93% resolution efficiency in water, per the American National Standard Institute IOL Optics Standard, revealed the IOL's excellent optics. This was confirmed by 0.278 wave root mean square surface figure as measured by Zygo interferometer using a 633 nm wavelength. Visual inspection revealed rough edges with sharp corners and some surface scratches. Early clinical experience with Ridley IOLs in Bombay, India, is briefly given. CONCLUSION: The Ridley IOL had excellent optical quality, meeting the requirements of current IOL optics standards. The selection of its dimensions was guided by the human crystalline lens, and the Ridley IOL was half as bulky. Although its clinical results were mixed, successful cases inspired subsequent improvements, leading to modern, highly satisfactory IOLs. This IOL represented a revolutionary innovation in ophthalmology.

England↗
PubMed1995

Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase.

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.

Amino Acid Sequence↗
PubMed1994

Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.

Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.

Base Sequence↗
PubMed1990

Dose equivalent response of a TLD badge--influence of body backscatter.

In personnel monitoring, operational quantities recommended by ICRU Publication No. 39 for photon radiation can be realized by calibrating dosimeters on a phantom and considering body backscatter photons by using established conversion factors. Personnel dosimeters used in this study are based on CaSO4:Dy Teflon thermoluminescence dosimeter discs (TLD) that have a highly photon energy-dependent response. Since body backscattered photons have lower energies than the incidence photons, methods for correcting for energy dependence of both the incident and body backscattered photons have to be developed. By using readouts of two TLD discs (one under a composite metal filter and the other without a metal filter) in an empirical relation valid at all energies, it is possible to correct for the effect of change in response from change in the photon energies. It was found that the new operational quantities recommended by ICRU could be estimated to within +/- 15% by a TLD badge design based on this method. Angular dependence limits for photons in accordance with the new international standards and a high beta dose-equivalent discrimination in the mixed fields of beta and low-energy x rays could also be achieved.

Equipment Design↗
PubMed1990

Risk factors for pneumonia after percutaneous endoscopic gastrostomy.

Percutaneous endoscopic gastrostomy (PEG) is currently a popular method of administering enteral feeding. Most of these patients are elderly, debilitated, and chronically ill. They are on a number of medications and have multiple diseases. With impaired consciousness and swallowing disability, these patients are prone to develop pneumonia. In order to identify possible risk factors, we followed 24 men who underwent PEG for the occurrence of pneumonia or until they died. We then analyzed the medical records of these patients for potential risk factors for pneumonia. The presence of esophagitis during PEG placement endoscopy and history of pneumonia prior to PEG were significant risk factors. Advanced age and cerebrovascular accident (CVA) tended to indicate a higher risk of pneumonia. Taking these risk factors into consideration may be beneficial in the management of such patients.

Age Factors↗
PubMed1990

Esophageal contribution to chest pain in patients with coronary artery disease.

We conducted a prospective study to determine the role of the esophagus in causing chest pain in patients with established CAD on optimum therapy. Thirty-two men with documented CAD who complained of frequent and usually daily retrosternal chest pain were evaluated. Following a standard esophageal manometry and acid perfusion test, simultaneous two-channel ambulatory Holter monitor and esophageal pH record tests were performed for 24 hours. Fifty-three episodes of chest pain were documented in 20 patients; 11 patients were free of pain. Of the 20 patients who complained of chest pains, 17 (85 percent) demonstrated at least one episode of PPR, defined as a drop in distal esophageal pH to less than 4 within ten minutes before or after the onset chest pain. Episodes of asymptomatic GER were common. The correlation of PPR with chest pain was 70 percent (37/53 episodes) and of ischemic ECG changes with chest pain 13 percent (7/53); in the remaining, there was no correlation with either. Two patients demonstrated simultaneous PPR and ischemic ECG changes. Seventeen esophageal motility abnormalities were observed in 14 patients (45 percent). It is our conclusion that esophageal disorders contribute to chest pain in patients with documented CAD. In this group, GER plays a greater role than in those with normal coronary arteries. In addition, esophageal motility disorders are common in these patients. Esophageal testing can be undertaken safely in these patients.

Aged↗
PubMed1989

The value of Chiba fine-needle aspiration biopsy in the diagnosis of hepatic malignancy: a comparison with Menghini needle biopsy.

The detection or exclusion of metastatic liver involvement is critical in the management and prognosis of patients with malignant disease. Noninvasive imaging modalities such as computed tomography, ultrasound, and technetium colloid liver scan are highly sensitive but nonspecific. Serum alkaline phosphatase is of similar value. A blind liver biopsy by the Menghini technique is often done to confirm the diagnosis, but its yield is low. We prospectively evaluated 74 patients using blind Menghini needle biopsy and concurrent Chiba fine-needle aspiration biopsy (FNAB) techniques. A positive diagnosis of malignancy was made in 30 patients (41%). In only 25 (34%) was the diagnosis made by Menghini biopsy, while Chiba FNAB confirmed the diagnosis in all 30 patients. Thus, concurrent use of both needles increased the diagnostic accuracy by 7%. Seven additional patients, considered to have one or more contraindications for the Menghini biopsy, underwent Chiba FNAB alone; the diagnosis was confirmed in all without complication. We conclude that FNAB alone or in combination with Menghini biopsy is valuable and safe in the diagnosis of metastatic liver disease.

Adult↗