Microgram Of DNA Research Articles

Transfection ofActinomycesspp. by Genomic DNA of Bacteriophages from Human Dental Plaque

Bacteriophages that produced turbid or clear zones of lysis in strains ofActinomyceswere isolated from 22 of 124 samples of fresh human dental plaque. All human and nonhuman strains ofActinomyces viscosusorActinomyces naeslundiitested in this study were sensitive to infection by one or more of these phages. In contrast, none of theActinomyces odontolyticus, Actinomyces israelii,orActinomyces bovisstrains tested were susceptible. Results of restriction endonuclease analyses indicated that the genomes of these phages consisted of double-stranded DNA molecules ranging in size between 16 and 60 kbp. Sequence homology under hybridization conditions of high stringency was observed among a few of the isolated phages. A lysogenized isolate ofA. viscosusMG-1 was obtained following infection with a temperate phage, designated φ225. Results of Southern blot analyses indicated that φ225 replicated as a plasmid in the lysogenized strain. Genomic DNA from several lytic phages was used to establish conditions for transfection by electroporation of strains ofActinomycesspp. Efficiencies of DNA transfer ranged from 102to 105plaque-forming units per microgram of DNA were obtained under optimal transfection conditions. The results of these studies demonstrate that transfer of genetic information inActinomycesspp. can be achieved by transfection.

Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery.

We have characterized the relationships between the design of cationic liposomes as a gene transfer vehicle, their resulting biodistribution and processing in animals, and the level and sites of gene expression they produce. By redesigning conventional cationic liposomes, incorporating cholesterol (chol) as the neutral lipid and preparing them as multilamellar vesicles (MLV), we increased the efficiency of cationic liposome:DNA complex (CLDC)-mediated gene delivery. Expression of the luciferase gene increased up to 1,740-fold and of the human granulocyte-colony stimulating factor (hG-CSF) gene up to 569-fold due to prolonged circulation time of injected CLDC, and increased uptake and retention in tissues. The level of gene expression per microgram of DNA taken up per tissue was 1,000-fold higher in lung than in liver, indicating that in addition to issues of delivery and retention of injected DNA, tissue-specific host factors also play a central role in determining the efficiency of expression. Vascular endothelial cells, monocytes, and macrophages are the cell types most commonly transfected by intravenous injection of CLDC.

Mitogenic and Metabolic Actions of Epidermal Growth Factor on Rat Articular Chondrocytes: Modulation by Fetal Calf Serum, Transforming Growth Factor-β, and Tyrphostin

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [3H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [35S]sulfate incorporation, and collagen synthesis by [3H]proline incorporation. The presence of specific receptors was confirmed by [125I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5–2.5 ng/ml) stimulated [3H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (≤1%) and was lost at high (≥7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-β and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites (Kd0.18 nm). There were about 4.5 × 109binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions.

Long-range amplification of genomic DNA detects the t(2;5)(p23;q35) in anaplastic large-cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis

Determine the frequency of t(2;5)(p23;q35) in anaplastic large-cell lymphoma (ALCL), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and lymphomatoid papulosis (LP). The t(2;5) was detected with a long-range nested polymerase chain reaction (PCR) using 0.5 microgram of DNA (60,000-80,000 cells), 5'-primers from the NPM gene, 3'-primers from the ALK gene, agarose electrophoresis, hybridization, and autoradiography. Patients were evaluable if a 3016 base pair amplicon could be generated from tumor DNA with beta-globin primers. Amplicons were detected by PCR of genomic DNA from three ALCL cell lines and four primary ALCLs known to t(2;5) positive. DNA from t(2;5)-positive cell lines diluted 10(4)-fold or 10(5)-fold generated amplicons in 100% or 20% of reactions, respectively. Archival tumor DNA from 144 patients was amplifiable by beta-globin amplicons in 126 (88%) who are considered evaluable for this study. Twenty-two had ALCL, 69 other NHLs, 30 HD, and five LP. Genomic DNA PCR detected the t(2;5) in 5 of 22 with ALCL (23%, 95% confidence intervals [95% CI] 8%-45%) but not in those with NHLs, HD, or LP. Among ALCLs the t(2;5) was confined to 5 of 20 with nodal presentations (25%, 95% CI 9%-49%), among whom it was seen in 5 of 15 with T-cell or null-cell phenotype (33%, 95% CI 12%-62%), in 4 of 11 with age < 40 years (36%, 95% CI 11%-69%), and in 4 of 9 with nodal presentations, T-cell or null-cell phenotype, and age < 40 years (44%, 95% CI 14%-79%). Amplicon sizes were different between cell lines and patients, reflecting unique genomic DNA breakpoints, as confirmed by DNA sequencing, and served as an internal control against specimen cross-contamination in the laboratory. Long-range PCR of genomic DNA detects t(2;5) only in ALCL, but not in other NHLs, HD, or LP. Long-range PCR may be useful in establishing diagnosis, determining prognosis, and monitoring minimal residual disease in ALCL.

Effect of Aprotinin on Smooth Muscle Cell Proliferation, Migration, and Extracellular Matrix Synthesis

Aprotinin (AP) is a plasmin inhibitor commonly used to limit bleeding during reoperative coronary bypass surgery. The effect on smooth muscle cell (SMC) behavior and the development of intimal hyperplasia has not been evaluated. This study examines the effect of AP on SMC proliferation, migration, and extracellular matrix synthesis. Bovine aortic SMCs (three to five passages) plated at 5 × 104cells/ml were treated with 1.0, 10, 100, and 1000 μg/ml AP. Proliferation was measured as micrograms of DNA per well, after 3 days in culture. SMC migration was measured after treatment with mitomycin C (20 μg/ml, to study migration in the absence of proliferation) by means of a fence assay. Extracellular matrix was quantitated utilizing a tritiated proline assay and corrected for cell density (cpm/μg DNA). Data are presented as means ± SEM; ANOVA and post hoc Tuckey test were used to test for significant differences. AP stimulated SMC proliferation at dosages of 100 μg/ml (353 ± 6 μg DNA, SMC control vs 440 ± 11, 100 μg/ml;P< 0.05) and 1000 μg/ml (353 ± 6, SMC control vs 503 ± 14, 1000 μg/ml;P< 0.01). AP concentrations less than 100 μg/ml had no effect on SMC proliferation. A dose–response relationship existed between AP and SMC migration. AP doses as low as 10 μg/ml increased SMC migration from 66 ± 4 mm2(control SMC) to 90 ± 4 mm2(P< 0.02) while the maximal dose of AP (1000 μg/ml) produced an almost twofold increase in migration (66 ± 4, SMC control vs 113 ± 3, 1000 μg/ml group;P< 0.0001). AP had no effect on extracellular matrix synthesis. AP stimulated vascular SMC migration and proliferation but not matrix synthesisin vitro.These data suggest that AP could augment the development of intimal hyperplasia following bypass surgery.

Expression of biglycan, decorin and fibromodulin in the hypertrophic phase of experimental osteoarthritis

This study sought to assess the relative levels of the mRNAs of the core proteins of the small proteoglycans (PGs) biglycan, decorin and fibromodulin in the hypertrophic phase of the early osteoarthritis (OA) that follows joint injury. Experimental OA was induced in eight dogs by transection of the anterior cruciate ligament. Articular cartilage was harvested from each joint, the total RNA was extracted and the concentration of DNA in the cartilage was measured. The relative levels of mRNA for biglycan, decorin and fibromodulin were assessed by northern blot analyses. An increase in cartilage mass with no increase in DNA concentration confirmed that the joints were in the hypertrophic phase that follows joint injury. The total RNA per microgram of DNA was increased 2.5 times. Compared with control cartilage, the mRNA levels in osteoarthritic cartilage, when normalized to the concentration of DNA, were increased 3.9 times for biglycan, 1.2 times for decorin and 2.4 times for fibromodulin. Because these small PGs affect collagen fibrillogenesis in vitro, their discoordinate metabolism may contribute to the abnormal collagen formation and deposition that occurs in OA and to the ultimate failure of the articular cartilage.

Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR.

A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 microgram of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of < or = 32 HHV-6 genomes/microgram DNA. The viral burden in the ninth individual was 1.2 x 10(6) HHV-6 genome copies/microgram DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/microgram DNA, range 0-43321) compared to controls (10 copies/microgram DNA, range 0-423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.

Development of a sensitive porcine granulosa cell bioassay for follicle-stimulating hormone (FSH).

Our objective was to develop a sensitive in vitro bioassay for follicle-stimulating hormone (FSH) that does not require the housing of animals in a research facility. Porcine granulosa cells from 1- to 3-mm follicles were cultured on laminin for 48 hr in serum-free medium in the absence or presence of FSH or with other purified pituitary hormones, supplemented with 19-OH androstenedione. Estradiol accumulation in medium per microgram of DNA of cells was determined as a reflection of FSH-induced aromatase activity. FSH (0.01-10 ng/ml) caused a dose-dependent increase in estradiol production per microgram of DNA, with 1, 10, and 100 ng/ml significantly higher than control. Porcine FSH was approximately two fold more biopotent than rat FSH in this system. Higher doses of FSH (100 ng/ml) caused less estradiol accumulation, presumably reflecting FSH receptor down regulation. No other pituitary hormone produced significant estradiol accumulation. Unextracted serum from a patient with premature ovarian failure (10-50 microliters) was tested in parallel to purified rat FSH (0-50 ng/ml) in this system, resulting in similar estradiol accumulation per microgram of DNA. We have developed a porcine granulosa cell bioassay for FSH which is sensitive, is specific for FSH, and does not require the housing of animals on site. It can be completed by a technician within 4 working days and can detect FSH in a sample of human serum.

Suppression of matrix metalloproteinase production by hCG in cultures of human luteinized granulosa cells as a model for gonadotrophin-induced luteal rescue.

Granulosa cells were isolated from follicular aspirates collected at ovum recovery for in vitro fertilization. Cells were cultured in a defined medium on artificial extracellular matrix (Matrigel) in the presence or absence of hCG as a model for corpus luteum function. Release of cells from this culture system is reduced by hCG and this effect may be mediated through an inhibition of extracellular matrix degradation. Using zymography and western blot analysis, we confirm the identity of matrix metalloproteinases-2 and -9 in culture media. Matrix metalloproteinase-9 was the predominant gelatinase in freshly prepared granulosa cells and in culture media, and also represented a major metalloproteinase component in homogenates of early and mid-luteal phase samples of corpora lutea. Quantitative analysis of matrix metalloproteinases in culture media, obtained throughout the 14 day culture period and expressed per microgram of DNA, showed that matrix metalloproteinase-2, undetectable on day 2, rose throughout the culture period and that this rise was significantly inhibited by hCG. In contrast, matrix metalloproteinase-9 was clearly detectable on day 2 and remained relatively constant throughout much of the culture (day 2 to day 12) in the presence of gonadotrophin. Significantly increased production of matrix metalloproteinase-9 (day 6 to day 12) was evident in the absence of hCG. Our results provide further evidence for the hypothesis that the rescue of the corpus luteum in early pregnancy involves the maintenance of cellular function through the stabilization of the extracellular matrix.

Polymerase Chain Reaction Amplification of Genetic Loci from Diseased Channel Catfish Found Dead in Ponds

Abstract As part of a selective breeding program for farm-raised channel catfish Ictalurus punctatus, we screened diseased fish to identify genetic markers linked to disease resistance or susceptibility. Because many diseased fish in ponds are not detected until after death, we investigated the utility of DNA isolated from diseased channel catfish found dead in ponds. Channel catfish (4–25 g) diagnosed with enteric septicemia of catfish or saprolegniasis were sampled 24–48 h postmortem from infected ponds. Control fish were killed by anesthetic overdose and sampled immediately. Total DNA isolated from liver, muscle (with skin), and caudal fin was quantified and analyzed for degradation. Yield of purified DNA, measured as micrograms of DNA per milligram of tissue, was significantly (P < 0.05) lower in diseased fish than in controls. Two sets of DNA primers were used to amplify a portion of the channel catfish growth hormone gene and the mitochondrial D-loop region with the polymerase chain reaction. Degrad...

An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance.

A transformation system has been developed for the pathogen fungus Botrytis cinerea, based on the utilization of the wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin. Transformed protoplasts were regenerated at 10-25 micrograms ml(-1) of phleomycin, at a frequency of 25-40 transformants per microgram of DNA, and they were resistant up to 50 micrograms ml(-1). Southern hybridization using undigested and digested total DNA showed the presence of circular autonomously replicating plasmid pUT737 in the transformants. Reisolated plasmid from transformed fungus transformed E. coli and rescued plasmid was identified as PUT737. Transformants were grown for four generations under non-selective conditions and replicative plasmids were still detected. Plasmids present in all transformants at this stage had been modified from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms has happened.

Quantification of 3'OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay.

A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules. The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA. After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules. Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of [32P]dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks). The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis. It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents. Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III. The assay is capable of quantifying first several breaks per 10(5) bp.

Excision of Oxidative Cytosine Modifications from γ-Irradiated DNA byEscherichia coliEndonuclease III and Human Whole-Cell Extracts

The possible release from γ-irradiated DNA of eight oxidatively modified cytosine bases byEscherichia coliendonuclease III was examined by trimethylsilylation and gas chromatography/electron impact/mass spectrometry. The results indicated that endonuclease III induced the release of 5-hydroxyhydantoin (1), 5-hydroxyuracil (2),cis-uracil 5,6-glycol (3), 5-hydroxycytosine (4),trans-uracil 5,6-glycol (5), andtrans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (8). The release of these products increased with the initial amount of damage in DNA, i.e., the dose of γ-radiation (0–100 Gy), giving 4.6 ± 1.0 fmol of 1, 5.8 ± 0.3 fmol of 2, 4.9 ± 0.5 fmol of 3, 11.2 ± 1.2 fmol of 4, 10.7 ± 2.1 fmol of 5, and 1.5 ± 0.5 fmol of 8, per microgram DNA per 10 Gy. In addition, we estimated that the relative rates of excision were 5 ≅ 3 > (1.2-fold) 1 > (1.5-fold) 4 > (3.3-fold) 2 on the basis of their initial yields in DNA and initial rates of release as a function of incubation time. The excision of 5-hydroxyuracil (2) and 5-hydroxycytosine (4) lesions was studied in greater detail by enzymatic digestion and HPLC coupled to electrochemical (EC) detection which determines the amounts of these products in DNA. The results showed that the excision of 4 was more efficient than that of 2 (2.7-fold) with greater than 50% of the lesions remaining in DNA after treatment. Finally, we examined the excision of products 2 and 4 from irradiated DNA (50 Gy) by whole human cell extracts. The release of product 2 into the hydrosylate was 5.2 ± 1.4 fmol per microgram of DNA as measured by fluorobenzylation coupled to gas chromatography/electron capture negative-ion chemical ionization/mass spectrometry. In identical samples, the amount of product 2 was reduced by 45.0 ± 2.6% (225 from 500 fmol per microgram of DNA) and that of product 4 by 7.0 ± 3.1% (42 from 600 fmol per microgram of DNA) as measured by HPLC/EC analysis.

Cationic lipid binding to DNA: characterization of complex formation.

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.

A homologous and self-replicating system for efficient transformation of Fusarium oxysporum.

A highly efficient transformation system has been developed for Fusarium oxysporum f. sp. lycopersici based on the complementation of a nitrate-reductase mutant with the homologous nit1 gene and on the presence of ARS and telomeric sequences in the vector. Preliminary transformation experiments with the niaD gene from Aspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with the F. oxysporum nit1 gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicative F. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per microgram of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment from F. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with the ARS from different organisms has shown that this fragment contained the commonly identified ARS consensus sequence, 5'A/TTTTATA/GTTTA/T3' and, in addition to this core, ten copies of the ARS-box, 5'TNTA/GAA3'. Adjacent to this presumed ARS, the telomeric hexanucleotide sequence (TTAGGG)n was present in six tandem copies followed by 18 copies of its complementary sequence.

Potentiation of cationic liposome-mediated gene delivery by polycations.

We discovered that several high molecular weight cationic polymers, such as poly(L-lysine) and protamine, can enhance the transfection efficiency of several types of cationic liposomes by 2-28-fold in a number of cell lines in vitro. Small polycations such as spermine and a cationic decapeptide derived from SV40 T-antigen were only moderately active. The addition of poly(L-lysine) and protamine dramatically reduced the particle size of the complex formed between DNA and cationic liposomes and rendered DNA resistant to the nuclease activity. The complexes composed of DNA, poly(L-lysine), and cationic lipids were purified from an excess of free liposomes with sucrose gradient ultracentrifugation. Purified complex formed at low cationic liposome ratio was poor in lipid content and only had weak transfection activity. Addition of free liposome to the purified complex significantly enhanced the transfection activity. In contrast, complexes formed at a higher initial ratio of liposome to DNA had a higher lipid content and were highly active in transfection; the activity was about 3-9-fold more active than the corresponding complex before purification. Negative stain EM studies revealed that the most active complexes prepared from 40 nmol of lipid, 0.5 micrograms of poly(L-lysine), and 1 microgram of DNA and purified by gradient ultracentrifugation were spherical, electron dense, small (< 100 nm in diameter) particles, and some of them were associated with lipid membranes. These highly active, stable, small-sized lipid/poly(L-lysine)/DNA complexes represent a new class of nonviral gene delivery vehicles that might be useful in gene therapy.

Isolation and characterization of a plasmid from Lactobacillus helveticus CP53.

A plasmid, pCP53 (11.5 kilo base pairs long), was isolated from Lactobacillus helveticus CP53 and its restriction map was constructed. A chimeric plasmid, pCP53D (4.7 kb), which had a 2.0-kb HindIII fragment from pCP53 and a tetracycline resistance (Tc) gene from Streptococcus faecalis replicated and expressed tetracycline resistance in Lactobacillus casei CP680. Fifteen strains of L. helveticus were transformed with pCP53D by electroporation and selected for Tc resistance. Transformants were obtained from two out of 15 host strains, L. helveticus CP611 and CP791, with 5 x 10(3) and 50 transformants per microgram of DNA, respectively.

Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.

A Semiquantitative PCR Assay for Assessing Perkinsus marinus Infections in the Eastern Oyster, Crassostrea virginica

A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using 1 microgram of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 microgram of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot-blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a dilution series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as "dilution endpoint" PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification of P. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat.