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S M Beverley

Publications and source records attributed to S M Beverley.

At least 109 records · Page 6Linked to original sources
PubMed1988

Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis.

Large circular amplified DNAs (30 and 85 kb) present in methotrexate-resistant Leishmania major appear to migrate anomalously in pulsed field-gradient electrophoresis (PFGE), exhibiting pulse time-dependent mobility and migrating along a different apparent path relative to the large linear chromosomal DNAs. Quantitative studies indicate that the relative pulse-time dependence is actually conferred by the mobility properties of the large linear DNAs. One contributing factor to the difference in migration path is variability in the intrinsic voltage-dependence of mobility of supercoiled and linear DNAs, in combination with the asymmetrical/inhomogeneous voltage gradients. Certain linear chromosomes exhibit a previously undescribed pulse-time dependence in the voltage-dependence of mobility. When enzymatically relaxed or physically nicked the large circular DNAs fail to leave the well using any pulse time, a property also observed in conventional electrophoresis. These findings are relevant to PFGE theory, and its application to the study of circular DNA amplification in Leishmania and other species.

Animals↗
PubMed1988

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

Animals↗
PubMed1987

Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification.

The methotrexate (MTX) and folate transport properties of five MTX-resistant lines of Leishmania major have been examined. These resistant lines all show a decreased Vmax for MTX influx, with no change in apparent affinity (Kt). The Vmax of folate influx is also proportionately decreased without alteration in Kt, supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two regions of DNA, the R region (encoding dihydrofolate reductase-thymidylate synthase) and the H region (Beverley, S.M., Coderre, J.A., Santi, D.V., and Schimke, R.T. (1984) Cell 38, 431-439), were also observed. In a given line, the amplifications occurred singly, in combination, or not at all. No other regions of amplification were detected. The phenotype of reduced MTX transport was moderately stable in the highly resistant R1000 line, being retained for more than 200 generations in the absence of MTX in vitro and during one passage through an infected mouse; in contrast, R- and H-amplified DNA were less stable. The lack of correlation of R and H amplification with reduced MTX transport suggests that alterations in transport are not causally mediated by gene amplification in Leishmania, but are a separate mode of MTX resistance. The onset of decreased MTX transport was also examined; wild-type Leishmania developed a reduced Vmax of MTX influx within 24 h following exposure to 1 microM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the Vmax of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As the folate/MTX transporter is regulated by exogenous folate, these data suggest that the initial rapid decrease in MTX transport may be a cellular regulatory response, in contrast to that found within the R1000 line which resembles a more stable genetic mutation.

Animals↗
PubMed1987

Biochemistry and regulation of folate and methotrexate transport in Leishmania major.

Promastigotes of the protozoan parasite Leishmania major exhibit high affinity uptake of folate (Kt = 0.7 microM) and methotrexate (MTX) (Kt = 1.8 microM) which is saturable and sensitive to metabolic poisons. Influx of folate and MTX is competitively inhibited by 5-formyltetrahydrofolate and p-aminobenzoic acid-glutamate, but not by 4-deoxy-4-amino-10-methylpteroate, biopterin, or pteroate. A single carrier is inferred for both folate and MTX transport, as the Ki of each inhibitor for both folate and MTX influx is the same, and the apparent affinities (Kt) of the substrates folate and MTX are identical to their respective Ki values for inhibition of MTX and folate uptake. Folate influx is specifically regulated according to cellular growth phase, as stationary phase cells exhibit 7% of the Vmax of log phase cells, while energy-dependent glucose uptake is only moderately reduced in stationary phase. Folate influx is also regulated by external folate levels, as cells grown in 5 microM folate exhibit 30% of the Vmax of cells grown in folate-depleted medium. Comparison of bacterial, mammalian, and Leishmania folate transport activities indicates considerable diversity in both biochemical and regulatory properties, and suggests the possibility that selective inhibition or manipulation of folate transport may be exploited in parasite chemotherapy.

4-Aminobenzoic Acid↗
PubMed1987

Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.

Animals↗
PubMed1987

Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns.

Restriction endonuclease DNA fragment patterns have been used to examine the relationships among 28 isolates of Leishmania as well as Crithidia, Endotrypanum, and Trypanosoma cruzi. Fragments of nuclear DNA were generated with six restriction enzymes, and blots were hybridized with probes from three loci. Among the major lineages the fragment patterns are essentially completely different, while within the major lineages various degrees of divergence are found. Molecular evolutionary trees were constructed using the method of Nei and Li to estimate the percent nucleotide sequence divergence among strains from the fraction of fragments shared. Defined groups, such as species or subspecies within the major lineages, are also grouped by nuclear DNA comparisons. Within the donovani complex, we find Leishmania donovani chagasi and Leishmania donovani infantum to be as similar as strains within Leishmania donovani donovani, consistent with the proposal by other workers that New World visceral leishmaniasis originated quite recently.

Animals↗
PubMed1987

In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes.

We examined the susceptibility of promastigotes of Leishmania major to sulfonamides and sulfones in vitro. In a completely defined medium only sulfamoxole, sulfaquinoxaline, and dapsone were inhibitory; the concentrations required for 50% inhibition of the rate of growth were 150, 600, and 600 microM, respectively. Eleven other sulfa drugs were ineffective at concentrations up to 2 mM. The growth inhibition was similar to that observed in procaryotes: the cells continued logarithmic growth for several cell doublings before inhibition was observed. Surprisingly, the addition of p-aminobenzoate or folate did not reverse the effects of the active sulfa drugs, the effects of sulfamoxole and methotrexate were additive rather than synergistic, and the addition of thymidine reversed methotrexate but not sulfa-drug inhibition. These results suggest that the mode of action of sulfa drugs on L. major is not by the classical route of inhibition of de novo folate synthesis. Promastigotes could be propagated for more than 40 passages in a completely defined medium in which the only added pterin was biopterin. The folate concentration in this medium was less than 10(-10) to 10(-11) M, as determined by a Leishmania bioassay. Although these data suggest that L. major may be capable of de novo synthesis of folate, the nonclassical mode of action of sulfa drugs, as well as other studies, favors the view that L. major is auxotrophic for folate.

Animals↗
PubMed1986

Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major.

We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and the rate of evolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.

Amino Acid Sequence↗
PubMed1985

Ancient origin for Hawaiian Drosophilinae inferred from protein comparisons.

Immunological comparisons of a larval hemolymph protein enabled us to build a tree relating major groups of drosophiline flies in Hawaii to one another and to continental flies. The tree agrees in topology with that based on internal anatomy. Relative rate tests suggest that evolution of hemolymph proteins has been about as fast in Hawaii as on continents. Since the absolute rate of evolution of hemolymph proteins in continental flies is known, one can erect an approximate time scale for Hawaiian fly evolution. According to this scale, the Hawaiian fly fauna stems from a colonist that landed on the archipelago about 42 million years ago-i.e., before any of the present islands harboring drosophilines formed. This date fits with the geological history of the archipelago, which has witnessed the sequential rise and erosion of many islands during the past 70 million years. We discuss the bearing of the molecular time scale on views about rates of organismal evolution in the Hawaiian flies.

Animals↗
PubMed1984

Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification.

We have employed the technique of chromosome "walking" to determine the structure of 240 kilobases of amplified DNA surrounding the dihydrofolate reductase gene in methotrexate-resistant mouse cell lines. Within this region, we have found numerous DNA rearrangements which occurred during the amplification process. DNA subclones from regions flanking the dihydrofolate reductase gene were also utilized as hybridization probes in other cell lines. Our results show that: 1) amplification-specific DNA rearrangements or junctions are unique to each cell line; 2) within a given cell line, multiple amplification-specific DNA sequence rearrangements are found; 3) the degree of amplification of sequences flanking the dihydrofolate reductase gene shows quantitative variation among and within cell lines; and 4) both the arrangement of amplified sequences as well as the magnitude of gene amplification may vary with prolonged culture even under maintenance selection conditions. These studies indicate that there is no static repetitive unit amplified in these cells. Rather, a dynamic and complex arrangement of the amplified sequences exists which is continually changing.

Animals↗
PubMed1984

Molecular evolution in Drosophila and the higher Diptera II. A time scale for fly evolution.

In this paper, we examine first the steadiness of the rate of evolutionary change in a larval hemolymph protein, LHP, in numerous Drosophila species. We estimated amino acid sequence divergence from immunological distances measured with the quantitative microcomplement fixation technique. Using tests not depending on knowledge of absolute times of divergence, we estimated the variance of the rate of evolutionary change to be at least 4 times as large as that for a process resembling radioactive decay. Thus, the rate of evolution of this protein is as uniform as that of vertebrate proteins. Our analysis indicates no acceleration of protein evolution in the lineages leading to Hawaiian drosophilines. Second, we give an explicit description of a procedure for calculating the absolute value of the mean rate of evolutionary change in this protein. This procedure is suggested for general use in calculating absolute rates of molecular evolution. The mean rate of evolution of LHP is about 1.2 immunological distance units per million years, which probably corresponds to a unit evolutionary period of 4 million years; LHP thus evolves at a rate comparable to that of mammalian hemoglobins. Finally, we utilize the calibrated rate of LHP evolution to derive a time scale of evolution in the Drosophilidae and higher Diptera.

Animals↗
PubMed1984

Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization.

Methotrexate-resistant Leishmania tropica contain two separate regions of DNA amplification, one encoding the bifunctional thymidylate synthetase-dihydrofolate reductase (TS-DHFR) characteristic of protozoans and the other of yet unknown function. The amplified DNAs are initially found as extrachromosomal closed circular forms, which are unstable in the absence of selection. After prolonged culture in methotrexate the amplified DNAs are found as repetitive arrays associated with the chromosomal DNA fraction after CsCl-ethidium bromide density gradient centrifugation, and are stable once selection is removed. The molecular description of gene amplification in Leishmania thus closely parallels the cytological features of gene amplification in cultured mammalian cells.

Animals↗
PubMed1984

A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa.

Thymidylate synthetase and dihydrofolate reductase exist as a bifunctional protein in a number of species of protozoa which span diverse groups of the subkingdom. The enzymes copurify upon gel filtration and on affinity chromatography columns specific for dihydrofolate reductase. The bifunctional protein has been found in species of Crithidia, Leishmania, Trypanosoma, Plasmodium, Eimeria, Tetrahymena and Euglena. For reasons unknown, neither enzyme could be detected in Entamoeba histolytica or E. invadens. Since neither enzyme has yet been found as a separate protein in protozoa, it is likely that the bifunctional protein is widespread among these primitive eukaryotes. In most cases, the apparent size of the native protein is approximately twice that of the subunit possessing thymidylate synthetase. Further, with one exception, the subunit sizes are close to the sum of the subunit sizes of the separate enzymes found in other sources.

Animals↗
PubMed1983

Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting.

We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 X 10(-2) amplification events per cell division) than do parental cells (ca. 10(-3) events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.

Animals↗
PubMed1983

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 kilobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.

Animals↗
PubMed1982

Molecular evolution of Drosophila and higher Diptera. I. Micro-complement fixation studies of a larval hemolymph protein.

LHP is a suitable protein for studying evolution in flies (Diptera). This blood protein, which occurs at high concentration late in larval development, was purified to homogeneity from 5 species of Drosophilidae and one species each of Tephritidae and Calliphoridae. Rabbit antisera to the purified LHPs allowed immunological comparisons to be made with the micro-complement fixation technique. Various indirect tests indicated that immunological distance is a reliable estimator of the degree of amino acid sequence difference between LHPs from different species. An evolutionary tree for the 7 LHPs was constructed from the immunological distances with the method of Fitch and Margoliash (1967) to provide the branching order and the method of Chakraborty (1977) to provide the branch lengths. A modified method of tree construction allowed LHPs from 10 additional species to be attached to this tree. The resulting LHP tree for 17 species agrees approximately in branching order with that based on Throckmorton's study of 60 anatomical traits. However, the ratio of anatomical change to LHP change along branches within the tree varies widely, confirming the independence of organismal and molecular evolution. The LHP tree thus provides a new perspective on evolution within and among the families of higher Diptera.

Animals↗
PubMed1981

Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts.

We have studied a line of 3T6 mouse embryo fibroblasts grown in progressively increasing concentrations of methotrexate. Resistance of cells to low concentrations of the inhibitor (less than 5 X 10(-5) M) is attributed to selective multiplication of the genes coding for dihydrofolate reductase and the resulting elevation of enzyme content. Cells isolated at a higher methotrexate concentration (4 X 10(-4) M) contain high levels of a dihydrofolate reductase with a reduced affinity for methotrexate. The altered dihydrofolate reductase exhibits a 270-fold reduction in binding affinity for methotrexate as measured by equilibrium dialysis (Kd = 5.4 X 10(-8) M versus 2 X 10(-10) M for the wild type enzyme). While binding to NADPH is unchanged, the Km for dihydrofolate is increased 3-fold over wild type enzyme and the turnover number for the reduction of dihydrofolate to tetrahydrofolate is decreased 20-fold. The altered dihydrofolate reductase shows a broader and more predominant acidic peak in its pH profile for this reaction. The molecular weights of the altered and wild type enzymes are identical as determined by sodium dodecyl sulfate-gel electrophoresis, but 2-dimensional electrophoresis reveals a significant basic shift in the migration of the altered enzyme. Studies with various folic acid analogs suggest that modifications involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases.

Animals↗