Cloning and overexpression of the gene encoding bacteriophage T5 DNA polymerase.

T5 DNA polymerase (T5Pol), an essential enzyme for bacteriophage T5 DNA replication, is unusual because of its high processivity and strand-displacing ability. These two properties in a single polypeptide make T5Pol an ideal candidate for structural and functional analysis. Therefore, the structural gene encoding the DNA polymerase of bacteriophage T5 (T5pol) has been cloned and overexpressed in Escherichia coli. Elimination of sequences upstream from the 5' end of the T5pol by exonuclease III digestion was necessary to obtain stable clones containing a full-length structural gene. Determination of the nucleotide (nt) sequence of the region deleted during clone construction revealed the presence of a promoter sequence having extensive homology with known T5 phage 'early' promoters. By primer extension of mRNA isolated from T5 phage-infected cells, two successive G residues located 6 and 7 nt downstream from the -10 region of this promoter were identified as the initiating nt at the 5' end of T5pol mRNA. T5Pol produced in E. coli from the cloned gene under control of a tac or phage lambda pL promoter represented as much as 40% of total cell protein. The majority of the T5Pol present in extracts of E. coli was insoluble. The amount of active enzyme present was estimated to be a maximum of tenfold higher than that found in extracts of T5 phage-infected cells.

Cocaine abuse and HIV-1 infection: epidemiology and neuropathogenesis.

The epidemiology of cocaine abuse and potential relationships of cocaine withdrawal to human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are discussed. Neuroendocrinological changes in HIV-1 infection of the central nervous system (CNS) are discussed with the relevant impact of cocaine abuse. HIV-1 load in the brain tissue of infected substance users is described along with possible associations with neuropathology and HAD. Finally, the molecular epidemiology and sequence heterogeneity of HIV-1 and their implications for neuropathogenesis are summarized. The complex context of addressing cocaine abuse in the setting of HIV-1 infection appears more tractable when decomposed into its components.

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Aging and neuro-AIDS conditions and the changing spectrum of HIV-1-associated morbidity and mortality.

Older individuals (>50 years of age) now comprise over 11% of patients with AIDS in the United States. This percentage is expected to continue to grow, due both to the improved longevity of patients prescribed highly active antiretroviral therapy (HAART) and to new infections among older individuals. This review focuses on the neuropsychiatric and neurological conditions that are most likely to be affected by advancing age-HIV-1-associated cognitive-motor disorder, peripheral neuropathy, progressive multifocal leukoencephalopathy, primary CNS lymphoma, and risk for cerebrovascular accident. Age associations with incidence of these disorders and with treatment foci are specified. Implications for future changes in management are discussed.

Polymerase chain reaction method for determining ratios of human immunodeficiency virus proviral DNA to cellular genomic DNA in brain tissues of HIV-infected patients.

A PCR method was developed to compare HIV-1 DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-1 provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (10-250 per 10(3) cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.

A rapid method for comparative quantitative polymerase chain reaction of HIV-1 proviral DNA extracted from cryopreserved brain tissues.

The quantitative polymerase chain reaction (PCR) method devised by Fujimura and Bockstahler (1995) was modified for rapid determination of distribution of HIV-1 proviral DNA load in AIDS brains. It was used for analysis of an association with HIV-1 associated dementia and HIV-1 encephalitis (Fujimura et al., 1997). The method has wider applicability for comparative studies of viral DNA load based on PCR amplification. The method is applicable under conditions where target DNA and its PCR-amplified product increase proportionally. An equation was derived to obtain the number of copies of HIV-1 DNA per cellular genome from the amount of PCR amplified product of a tissue specimen DNA. The equalizing constant is the reciprocal of the slope of the amplification of the HIV-1 proviral DNA sequence of the standard cellular DNA included in each experiment. The intercept of the equation is zero.

HIV-1 RNA load in needles/syringes from shooting galleries in Miami: a preliminary laboratory report.

We quantified HIV-1 RNA load in rinses from needles/syringes (N/S) obtained at shooting galleries in Miami and also analyzed the rinses for antibodies for viral proteins. In rinses from 36 N/S that contained visible blood, 14 (39%) had detectable amounts of HIV-1 RNA. Numbers of copies of HIV-1 RNA ranged from the detection limit (400 copies/ml) to 268,000 copies/ml. We also detected antibodies to HIV-1 polypeptides in 34/36 (94%) of rinses from visibly contaminated N/S using Western blots specific for the HIV-1 proteins. No antibodies were detected in laboratory rinses from six visibly clean needles. The presence of HIV-1 RNA in N/S is an important indication of the risk created by N/S sharing as well as by shared paraphernalia and wash waters by injecting drug users.

DNA as a solar dosimeter in the ocean.

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.

Dementia and the Neurovirulence of HIV-1.

Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.

Subtle neuropsychological impairment and minor cognitive-motor disorder in HIV-1 infection. Neuroradiological, neurophysiological, neuroimmunological, and virological correlates.

HIV-1 infection of brain may be associated with multiple treatment targets, only the most severe of which is represented by HAD. Focusing on earlier treatment targets such as MCMD and cognitive-motor impairment in the absence of any clinical disorder (as well as neuroprotection) may prove to be of greater clinical utility in the treatment and prevention of such impairment than a focus on later-stage cognitive-motor disease, when neuronal cell death is already extensive. This may be especially important now that improvements using the protease inhibitors in triple-drug combination regimens have reduced plasma viral load to unmeasurable levels, while these drugs do not penetrate the CSF well. Currently, peripheral blood markers do not appear to be highly sensitive for central nervous system impairment, and specific CSF laboratory markers have some limited value at present-while requiring a lumbar puncture to obtain. Hence, a role for noninvasive techniques using neuroimaging exists in the clinical management of HIV-1-infected patients. To date, structural imaging techniques have proven limited in value for HIV-1-specific impairment. Several functional techniques (PET, SPECT, and MR spectroscopy) have now provided promising results for the purposes of identifying clinically significant dysfunction, relating such dysfunction to clinical neuropsychiatric symptom status, and for treatment response monitoring. Further studies are needed to examine the extent to which such imaging modalities not only parallel clinically relevant aspects of HIV-1 disease progression, but also match specific types of neuropsychologic performance deficits with potential significance for neuroanatomical localization. It is particularly important to include neurophysiological, neuroimmunological, and virological measures in studies that examine clinical neuropsychiatric status with neuroimaging techniques. In addition, the inclusion of neuropathology data, where possible, is important because demonstration of HIV-1 encephalitis cannot be equated with clinical disorder and because specific HIV-1-associated pathological changes have not yet been proven to be assessed well with neuroimaging techniques (e.g., the extent of microglial cell and macrophage activation). Also, treatment response studies are needed in conjunction with primary antiretroviral therapy regimens specifically aimed at central nervous system penetration (e.g., GW1592, GW141, and nevirapine). The results of such work will provide the data required to determine whether these promising functional neuroimaging techniques will aid in meeting the expected, imminent increase in clinical burden of this frequent complication of HIV-1 infection.

Processiveness of DNA polymerases. A comparative study using a simple procedure.

In this communication, we describe a simple procedure for analyzing the processiveness of DNA polymerases in general. By choosing conditions for which the number of incorporations per available primer is less than 1, we have reduced the probability of a primer molecule being utilized by the enzyme more than once. The primer-template used was poly(dA)300:oligo(dT)10, and the product was isolated by oligo(dT)-cellulose chromatography. The number of dTMP residues added per association was determined from the [3H]dThd + [3'-3H]dTMP/[3H]dThd ratio of the product after its digestion by micrococcal nuclease and spleen phosphodiesterase. Using this procedure, we have found that Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus alpha- and beta-DNA polymerase are "quasi-processive." Most of these enzymes add on the average approximately 10 to 15 nucleotides before dissociating from the template. T5 DNA polymerase, on the other hand, is processive, i.e. it continues to replicate a given template until it is very close to the 5' end of the template. With "nicked DNA-like" poly(dA):oligo(dT), the processiveness of E. coli DNA polymerase I is increased 2- to 2.5-fold. The significance of this increase in determining the "patch size" during DNA repair is discussed.

Temperature-sensitive DNA polymerase induced by a bacteriophage T5 mutant: relationship between polymerase and exonuclease activities.

DNA polymerase induced by bacteriophage T5ts53, a mutant with temperature-sensitive polymerase, was purified to about 95% purity as judged by dodecyl sulfate gel electrophoresis. The 3' leads to 5' exonuclease associated with the polymerase had higher activity than that associated with the parent wild-type enzyme. It was more stable to heat than the polymerase, and it degraded primer-template even in the presence of 4 dNTP's at higher temperature. However, the evidence presented shows that the inhibition of DNA synthesis by higher temperature was primarily due to defects in polymerase function rather than to overactive exonuclease. The presence of primer-template DNA stabilized the polymerase to heat. Purified ts53 polymerase was also shown to discriminate against incorportion of BrdUMP, especially at higher temperature. This is an agreement with observations made in vivo with ts53-infected bacteria.