Search PubMed⌕ Search

Biomedical subjects

J Griffith

Publications and source records attributed to J Griffith.

At least 163 records · Page 9Linked to original sources
PubMed1987

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

DNA, Bacterial↗
PubMed1986

Visualization of the bent helix in kinetoplast DNA by electron microscopy.

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.

Animals↗
PubMed1986

A gross morphologic, histologic, hematologic, and blood chemistry study of adult and neonatal mice chronically exposed to high magnetic fields.

Six sets of Charles River CD-1 mice (3 sets of adults and 3 sets of offspring: 156 mice total) were raised for varying times (360 h over 1 month to 624 h over 3 months) in a 1.89-T magnetic field. Each set was divided into 3 groups: control group raised in the animal facility (Control); control group raised in the magnet room but not in the magnet (Magnet-Controls); experimental group raised in the magnet (Magnet). At the end of each predefined exposure period, the mice were euthanized and studied. Body and organ weights were lower in 3 of 6 sets in the Magnet groups when compared to Control groups, but they were not significantly different from age matched, sex matched Magnet-Controls. This seems to indicate that the decreased growth of mice was due to the laboratory environment surrounding the magnet, when compared to the animal facility environment, rather than to magnetic field effects. No consistent differences were found in gross and microscopic morphology, hematocrit and white blood cell counts, plasma creatine phosphokinase, lactic dehydrogenase, cholesterol, triglyceride, or protein concentrations in Magnet groups compared to the two control groups.

Animals↗
PubMed1986

RecA protein filaments can juxtapose DNA ends: an activity that may reflect a function in DNA repair.

To further characterize the role of RecA protein-DNA filaments in general recombination and DNA repair, we have examined interactions of these filaments with themselves following formation. When linear double-stranded DNA was incubated with RecA in the presence of Mg2+ and adenosine 5'-[gamma-thio]triphosphate, monomer-length (1n) nucleoprotein filaments were observed. Following continued incubation, filaments having 2n, 3n, ... lengths were observed, indicating that an end-to-end joining of the monomer-length filaments had occurred. When linear single-stranded DNA was covered by RecA protein under several conditions, the ends of the resulting filaments joined together rapidly, producing circular filaments. The end-to-end joining of single-stranded DNA-RecA filaments appeared to require that 3' DNA ends be juxtaposed with 5' DNA ends, because double-stranded DNA molecules having long single-stranded DNA tails with only 3' or 5' termini did not join end-to-end. However, when both 5' and 3' ends were present in the reaction, joining was observed. We suggest that this end-to-end joining activity may help explain the role of RecA protein in both the protection of damaged DNA ends and the repair of double-stranded DNA breaks.

DNA Repair↗
PubMed1986

Visualization of the paranemic joining of homologous DNA molecules catalyzed by the RecA protein of Escherichia coli.

In reactions catalyzed by the RecA protein of Escherichia coli, synapsis between two DNA molecules is believed to occur even in the absence of free homologous DNA ends and to involve a metastable interaction termed paranemic joining. We have used electron microscopic methods to visualize synapse formation between supertwisted M13 double-stranded DNA (dsDNA) and linear M13 mp7 single-stranded DNA (ssDNA) with non-M13 sequences at its ends. These non-M13 sequences block strand invasion and make this pairing equivalent to the joining of two fully circular molecules. We observed a high frequency of joining when the ssDNA was initially assembled into presynaptic filaments with RecA protein. Cleavage of the dsDNA in the joined complexes by Hpa I revealed that the joint was at a site of homology. In these joints, the dsDNA entered the presynaptic filament over a length of 360 +/- 80 base pairs, not visibly altering its ultrastructure, and then dissociated from the filament. Although the dsDNA in the complexes appeared topologically relaxed, deproteinization released supertwisted dsDNA, indicating that the dsDNA was unwound by 34 degrees per base pair in the paranemic joint. When supertwisted M13 dsDNA was paired with circular M13 ssDNA, similar joints were observed and both DNA circles appeared topologically relaxed.

DNA, Single-Stranded↗
PubMed1986

Comparison of plasma cholinesterase depression among workers occupationally exposed to organophosphorus pesticides as reported by various studies.

A number of studies have reported on the inhibitory effects of organophosphorus pesticides (OPs) on the enzyme cholinesterase (ChE) among agricultural workers. With the increasing use of OPs, surveys of blood ChE activity on exposed workers may help to identify workers at greatest risk and to provide insight into the use history--e.g., mixing, loading, application, and harvesting--that might lead to a hazardous situation. Although it does appear that measurements of ChE activity are valuable in worker surveillance programs, it is difficult to interpret findings from various studies since they are dependent on the method of assay and the emphasis is usually placed on statistical tests (i.e., p values) that depend on the number of subjects studied. In the present paper a method is presented to compare ChE values reported in several studies utilizing various methods and units of measurement, and to assess the impact of OP exposure as a percentage of subjects with ChE values depressed below normal limits.

Agriculture↗
PubMed1985

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange.

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.

DNA, Circular↗
PubMed1985

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments.

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.

Adenosine Triphosphatases↗
PubMed1985

RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization.

The RecA protein of Escherichia coli, whether pure or in a crude cell lysate, will rapidly form small crystals (microcrystals) in the presence of low concentrations of spermidine. We describe the conditions of time, pH, and polyamine concentration over which crystallization occurs. Microcrystal formation is inhibited by concentrations of chloride over 25 mM and concentrations of phosphate or sulfate ions as low as 2 mM. Crystallization is not inhibited by high concentrations of other proteins, and the RecA protein microcrystals are easily collected by brief centrifugation. This provides a powerful purification step with high yield. Using this novel property, we prepared over 200 mg of RecA protein at least 95% pure with a single-strand DNA-dependent ATPase activity of 98% from 65 g of cells in 2-3 days. Spermidine was easily removed from the RecA protein by dialysis.

Crystallization↗
PubMed1985

10 nm RecA protein filaments formed in the presence of Mg2+ and ATP gamma S may contain RNA.

Filaments formed by the polymerization of RecA protein along DNA in the presence of Mg2+ and adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) are seen by electron microscopy to have a 10 nm diameter with a 9 nm helical repeat. When certain preparations of apparently pure RecA protein are incubated with Mg2+ and ATP gamma S in the absence of nucleic acid for extended times, very long filaments with the same 10 nm diameter and 9 nm axial repeat are seen. We show here that these long 10 nm filaments can contain RNA which is present as a contaminant of the RecA protein and poly(A) which is synthesized during the incubations by an activity that is apparently polynucleotide phosphorylase. RecA protein purified by a procedure developed in this laboratory did not contain RNA and did not form these very long 10 nm filaments. However, when exogenous RNA was added to this protein, 10 nm filament formation was observed.

Adenosine Triphosphate↗
PubMed1985

A community survey of psychological impairment among Anglo- and Mexican Americans and its relationship to service utilization.

Discussions of Mexican American mental health need have been limited to analyses of service utilization statistics, most of which have shown Mexican Americans to use mental health services proportionally less than other racial/ethnic groups. Consequently, Mexican Americans have been viewed as a group having greater unmet mental health needs. Yet, these utilization-based needs assessments make an implicit and untested assumption: that the proportion of the psychologically impaired is comparable across racial/ethnic groups. The purpose of the present investigation was, first, to contribute to a baseline measurement of psychological impairment among Mexican Americans in order to assess what utilization should be for this group and, second, to determine relationships between impairment symptomatology and sociodemographic characteristics which are unique to the Mexican American population. Anglo- and Mexican Americans (n = 515) living in one of three suburban communities in Southern California were selected by way of a random-digit, multistage cluster telephone sampling procedure. Respondents gave responses to a standardized measurement of psychological impairment. Results showed that Anglo- and Mexican Americans did not differ in anxiety, psychosocial dysfunction, and depressive symptoms. Mexican Americans in the three communities, however, were found to use mental health services proportionally less than Anglos. Relationships between age and psychosocial dysfunction symptoms, and between income and anxiety problems were different for Mexican Americans than those for Anglos. Issues related to what constitutes mental health need and the use of self-report measures of impairment to estimate mental health need across culturally diverse groups are discussed.

Adult↗
PubMed1985

Association of M13 I-forms and spheroids with lipid vesicles.

Electron microscopy and density gradient centrifugation were used to demonstrate that the coat protein of M13 I-forms and spheroids, but not of filaments, can form some type of association with lipid vesicles in vitro. The association was detected only when the phage particles were incubated with dilauroylphosphatidylcholine (DLPC) or dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) above the phase transition temperature of the lipid. Under these conditions the I-form coat protein was resistant to proteolytic digestion, and the viral DNA was also associated with the vesicles.

Capsid↗
PubMed1985

Grower reported pesticide poisonings among Florida citrus fieldworkers.

In a 1981 survey of 436 Florida citrus growers, 27 pesticide related poisoning incidents were reported that were to have taken place within one year of the interview date. From these reports it is possible to estimate that there are 376 citrus fieldworker related poisonings per year in Florida. This number of estimated poisonings may be developed into an incidence rate of 113 fieldworker poisonings per 10,000 fieldworkers.

Agricultural Workers' Diseases↗
PubMed1985

Pesticide poisonings reported by Florida citrus fieldworkers.

In a 1981 survey of 1811 Florida citrus fieldworkers, 25 pesticide related poisoning incidents involving 29 fieldworkers were reported. Suspected poisonings were categorized into possible and confirmed poisonings, and from these reports it was possible to project an estimated 438 possible poisonings, and 73 confirmed poisonings among all citrus fieldworkers. Confirmed pesticide poisonings were developed into an incidence rate of 34 poisonings per 10,000 permanent and semi-permanent fieldworkers. The number of possible and confirmed poisonings, for all fieldworkers, was then developed into an incidence rate of 160 poisonings per 10,000 fieldworkers.

Adolescent↗
PubMed1985

Monitoring study of urinary metabolites and selected symptomatology among Florida citrus workers.

A cross-sectional monitoring study was made of citrus fieldworkers employed during an entire citrus growing season in Florida. A survey questionnaire was administered to 1811 fieldworkers employed as applicators, mixers, loaders, tractor drivers, general combination workers, and pickers on 436 citrus groves throughout Florida. The study was designed to evaluate exposure to organophosphorus pesticides by analyzing the urine of citrus fieldworkers for dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), dimethyl dithiophosphate (DMDTP), diethyl phosphate (DEP), diethyl thiophosphate (DEPT), and diethyl dithiophosphate (DEDTP), and to relate the findings to the reported occurrence of selected health symptoms associated with pesticide intoxication. Urine samples were collected for analysis from 332 spray-season workers and 265 harvest-season workers. Measurable DEP residue values were found in 68% of the spray season workers and 43% of the harvesters. However, with the exception of DEP and DETP values in the urine of spray-season workers, metabolite residues were low, often at, or just above, detection limits (which were usually 0.02 ppm but occasionally rose higher, with a few at 0.03 ppm and a very few at 0.04 ppm). Although there was clearly exposure to organophosphates among the citrus fieldworkers, there was no apparent association between the reported health symptoms and the relatively low levels of organophosphate metabolites found in the urine of the workers.

Agricultural Workers' Diseases↗