Targeted gene knockout mediated by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Antioxidant, antioedema and analgesic activities of Andrographis paniculata extracts and their active constituent andrographolide.

Andrographis paniculata (AP), a popular ingredient of Oriental folk medicine, is commonly used for treating infection, inflammation, fever and diarrhoea. In this study, extracts prepared from cultivated AP and their active constituent andrographolide were evaluated for antioxidant, antioedema and analgesic activities. The results showed that the aqueous AP extract (AP-H2O) exhibited a greater antioxidant activity than the ethanol AP extract (AP-EtOH) in all model systems tested. At a concentration of 50 microg/mL, the free radical scavenging, xanthine oxidase inhibition and antilipid peroxidation activities for AP-H2O were 66.8%, 57.3% and 65.3%, respectively, and for AP-EtOH were 57.8%, 52.6% and 34.2%, respectively. At a dosage of 100 mg/kg, AP-H2O and andrographolide, but not AP-EtOH, showed antioedema and analgesic activities. In phytochemical analysis, AP-H2O showed a higher concentration of total flavanoid but a lower phenol content than AP-EtOH. In conclusion, AP-H2O was more potent than AP-EtOH in antioxidant activities. Furthermore, compared with andrographolide, AP-H2O as an extract also appears to possess potent antioedema and analgesic activities.

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'----3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'----5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk delta 5' and tk delta 3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'----3' degradative model and the other would be the favored substrate in the alternative 3'----5' degradative model. Our results indicate that the intermediate favored by the 3'----5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.

Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, can be crosslinked to 30 S ribosomal protein when the tRNA is placed at the ribosomal A site. This protein has now been identified by immunological methods. The protein-[3H]Phe-tRNA covalent complex, obtained by extraction with 6 M-urea, was reacted separately with each of the 21 purified antisera to 30 S ribosomal proteins. The double antibody technique was used. Anti-S19 was the only antiserum able to precipitate the radioactivity, and 66 to 81% of the added radioactivity could be precipitated. The same result was obtained with three different ribosome preparations, at low as well as high crosslinking yield, with dipeptidyl-tRNA in the A site as well as aminoacyl-tRNA, and when binding and crosslinking were performed at 20 mM-Mg2+ instead of at 5 mM. Therefore, when aminoacyl-tRNA or peptidyl-tRNA is in the ribosomal A site, position 8, which is always uridine or 4-thiouridine, must be within 9 A of protein S19.

Covalent crosslinking of Escherichia coli phenylalanyl-tRNA and valyl-tRNA to the ribosomal A site via photoaffinity probes attached to the 4-thiouridine residue.

tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.

Construct validity of a work environment impact scale.

This study examined the construct validity and the internal consistency of a newly developed assessment, the Work Environment Impact Scale (WEIS). After administration to 20 individuals with psychiatric disabilities, Rasch analysis was utilized to scrutinize the data. For this study, criteria for determining unexpected person/item responses were based on the following criteria: MNSQ > 1.3 and $\underline{t}>2.0$. Persons/items with MNSQ < 0.7 and $\underline{t}<-2.0$ were examined to enhance instrument precision but were not considered misfit. Results suggest that the WEIS is an appropriate and valid instrument to utilize with workers who have psychiatric disabilities. Overall, the items appeared to match the worker's need for performance, satisfaction, and well-being as the mean of persons measure is 0.30 ± 0.49 logits more than the mean of items measured. In addition the hierarchical order of items is consistent with literature identifying environmental press and affordance for workers with psychiatric disabilities. WEIS items constitute a uni-dimensional construct given that the summary statistics for both item and person had a MNSQ of 1.00 and 1.02 respectively and both $\underline{t}$ values were -0.2. However, three items exceeded the established criterion for being too informative given the MNSQ < 0.7 and a $\underline{t}<-2.0$., suggesting these needed to be revised to enhance the quality measurement of the instrument. One hundred percent of the workers fit the expected response pattern of the Rasch model suggesting that workers with greater satisfaction, performance and health had a higher degree of match with his/her occupational environment. In summary, anecdotal data suggested that the WEIS provided clinically relevant information useful for planning of work-related interventions or reasonable accommodations.

Pulmonary function studies in healthy Filipino adults residing in the United States.

BACKGROUND: Differences in lung volumes among various ethnic groups are known to occur; however, this has not been studied in Filipinos. OBJECTIVE: We sought to assess pulmonary function in healthy, nonsmoking Filipinos residing in the United States compared with standards for white subjects. METHODS: Healthy adult Filipinos, age 18 years or greater, were recruited. All subjects were screened with health questionnaires to exclude those with cardiopulmonary disease. Pulmonary function tests were performed by using forced expiratory maneuvers. Values for FEV(1 ), forced vital capacity (FVC), FEV(1 )/FVC, forced expiratory flow from 25% to 75% of FVC, and peak expiratory flow rate were compared with predicted values for white subjects (ie, without a racial adjustment). RESULTS: Two hundred twenty-four healthy subjects (121 men and 103 women) completed the study. The group means (as a percentage of the predicted standard for white subjects) were as follows: FEV(1 ), 86%; FVC, 84%; FEV(1 )/FVC, 103%; forced expiratory flow from 25% to 75% of FVC, 96%; and peak expiratory flow rate, 107%. These findings are very similar to those for African Americans and other Asians. CONCLUSION: We conclude that it is appropriate to use an 85% racial adjustment for FEV(1 ) and FVC when interpreting pulmonary function test results in Filipinos.

Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences.

In this paper, we show that DNA added to mouse L cells by the calcium phosphate method can be inserted into the genome of those cells by homologous recombination. The insertion event is detected because it reconstructs a functional thymidine kinase (tk) gene from two defective genes that share 320 base pairs of homology. One of the genes is missing its 5' portion (tk delta 5') and is in the cell's chromosome, and the other is missing its 3' portion (tk delta 3') and is in the introduced DNA. Gene reconstruction by homologous insertion is relatively inefficient; approximately one Tk+ transformant is produced per 10(6) cells per 4 micrograms of added tk DNA, a frequency of about 10(-5) that of normal tk gene transformation. The Tk+ transformants produced by homologous recombination contain Sma I and Pvu II fragments that are diagnostic of the intact tk gene, contain a herpesvirus-specific thymidine kinase activity, and can transfer the Tk+ phenotype to Tk- cells by DNA-mediated gene transfer. Two surprising observations made in the course of these studies were that only 1 of 10 Tk- cell lines containing defective tk genes could be transformed to Tk+ by homologous insertion of the complementary defective tk gene and that relatively little illegitimate insertion of introduced tk DNA into cellular DNA was detected in those cells that were transformed to Tk+ by homologous recombination.

Hypereosinophilia, neurologic, and gastrointestinal symptoms after bee-pollen ingestion.

A patient developed hypereosinophilia (13,440 cells per cubic millimeter) 6 weeks after beginning the ingestion of bee pollen. Symptoms included generalized malaise, headache, nausea, abdominal pain diarrhea, generalized pruritus, and decreased memory. Evaluation revealed no other known cause for the patient's hypereosinophilia, which resolved after bee-pollen ingestion was stopped. The product contained a mixture of entomophilous and anemophilous pollens to which the patient was skin test positive. An open challenge with the bee pollen later reproduced the presenting symptoms with a concomitant rise of the eosinophil count from 207 to 890 cells per cubic millimeter. The patient has since remained well avoiding bee pollen. This study strongly suggests that hypereosinophilia with attendant pathophysiologic disturbances may be an adverse reaction to bee-pollen ingestion in atopic individuals.

Are theophylline "levels" a reliable indicator of compliance?

Clinicians frequently rely on serum theophylline concentrations (STCs) as an indicator of compliance for asthma medications. Most patients with good compliance do not have excessive fluctuations during routine STC monitoring. However, our experience is that in certain patients, persistently low or erratic STCs may be a sign of abnormal theophylline disposition. With careful analysis of theophylline absorption (STC every 2 hours for 24 hours after oral theophylline doses) and elimination (serial STC for 12 hours after an intravenous dose of aminophylline), we identified several patients with previously unrecognized anomalies of theophylline pharmacokinetics. These include (1) a 16-year-old girl with consistent temporal fluctuation in STCs during administration of a sustained-release formulation every 8 hours because of delayed absorption and enhanced elimination of theophylline at night, (2) a 13-year-old girl with markedly delayed absorption of a once-daily preparation administered in the evening, (3) a 5-year-old boy with erratic absorption of a liquid theophylline preparation with significantly increased STCs during the night, and (4) a 49-year-old man with 60% bioavailability of aminophylline tablets. Based on these observations, we suggest that clinicians carefully consider the possibility of abnormalities in theophylline disposition before assuming unexpected deviations in STCs are solely the result of noncompliance.

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells.

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.