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The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (β-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a 'helical bouquet' with a 'ribbon' helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.

We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.

beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in single-stranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K(d) = 40-140 nm) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNA-binding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.

Modified DNA bases are widespread in nature. They can be found in eukaryotes, prokaryotes, and bacteriophages. They may completely replace the standard base or replace only a small fraction. Their substituents vary from simple methyl or hydroxy groups to large moieties like amino acids and multiply hexosylated side chains. This review gives an overview of the modified DNA bases identified thus far, with emphasis on the "very unusual" or hypermodified DNA bases. Although these have been detected mainly in bacteriophages, recent work has shown the presence of a novel hypermodified DNA base in the eukaryote Trypanosoma brucei. We speculate on the biosynthesis and function of this novel base beta-D-glucosyl-hydroxymethyluracil.

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture from were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1,221-246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.

Maxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by electron microscopy. Long loops up to 10 micrometer were present, extending from the network rim; these are considered typical of maxi-circles. The presence of maxi-circles was proven by digestion of kinetoplast DNA with restriction endonucleases and S1 nuclease. This released a minor DNA component, detectable by agarose gel electrophoresis, which hybridized to maxi-circle DNA from Trypanosoma brucei. The molecular weight of the linearized maxi-circle of Trypanosoma cruzi is 26 . 10(6), as judged from its electrophoretic mobility in 0.6% agarose. Our restriction enzyme analysis of the mini-circles of Trypanosoma cruzi has confirmed their sequence heterogeneity and internally-repeated structure. We have found that more than 90% of the mini-circles are cut into 1/4 length molecules by endonuclease TaqI. Denaturation and renaturation of mini-circles, cut once with endonuclease MboI, mainly yields linear and circular molecules with single-stranded eyes and tails in electron micrographs. This shows that 1/4 repeats contain sub-segments in which sequence divergence is extensive. Our EcoRI and HapII digests differ in fragment size distribution from those previously reported. This suggests that this distribution may not be a stable characteristic of the Y strain.

We have compared a total of 44 recognition sites for 12 restriction endonucleases on the 20 kilobase pair maxi-circle of kinetoplast DNA from nine Trypanosoma brucei stocks, four which are known to be infective to man (tow 'gambiense' and two 'rhodesiense' variants). In addition to five polymorphic sites, these DNAs differ in the size of a 5 kilo-base pair region which is cleaved only by one of the restriction enzymes tested and which varies in size over 1.5 kilo-base pairs. Our analysis shows that the maxi-circle sequences of these stocks are very similar, the maximal calculated difference between any two being 3%. A relatively large difference was found between a rhodesiense stock from uganda and one from Zambia, confirming the distinction between northern and southern East African rhodesiense stocks found by analysis of enzyme polymorphisms (Gibson et al. (1980) Adv. Parasitol. 18, 175-246). The gambiense variants could not be identified by unique restriction site polymorphisms, but contained the smallest maxi-circle found thus far in T. Brucei. Our results indicate that T. brucei stocks infective and not infective to man are so closely related as to preclude their differentiation by analysis of kinetoplast DNA. This analysis is useful, however, in providing quantitative information about relatedness of stocks.

Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.

We have studied the interaction of poly(rA) and poly(rU) with natural DNAs containing (dA.dT)n sequences. The results indicate that hybridization of poly(rA) to denatured DNA can be used to estimate the size and frequency of large (dA.dT)n tracts, whereas hybridization with poly(rU) does not give reliable information on these points. In 6.6 M CsCl, poly(rU) can form stable complexes with denatured DNA containing short (dA)n tracts (n less than or equal to 6), whereas binding of poly(rA) to denatured DNA under these conditions requires much larger (dT)n tracts (estimated n greater than 13). Moreover, binding of poly(rA) requires pre-hybridization in low salt, because free poly(rA) precipitates in 6.6 M CsCl.

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.

Previous work by Jacobson et al. (1) has shown that the number and distribution of (dA.dT)25 tracts in the nuclear DNA of the slime mold Dictyostelium discoideum reflects the number and the distribution of transcriptional units. To investigate whether this is a general phenomenon we compared the nuclear DNAs of other primitive eukaryotes with respect to their content of large (dA.dT)n tracts via the thermal stability of their hybrids with poly(rA). The results of our analysis indicate that all nuclear DNAs tested have (dA.dT)20-25 tracts, but the frequency of such tracts varies from one per 5.4 X 10(6) daltons in Dictyostelium nuclear DNA to one per 2.8 X 10(8) daltons in Crithidia luciliae nuclear DNA. We conclude that the presence of (dA.dT)20-25 tracts is not an obligatory characteristic of the transcriptional unit in primitive eukaryotes. Chromatography of native DNAs on poly(rU) Sephadex columns shows that the large (dA.dT)n tracts occurring in the genomes of both primitive and higher eukaryotes are widely distributed throught these genomes.

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.

Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.

The genome of Trypanosoma brucei carries over a hundred genes coding for different variants of the major surface glycoprotein. Activation of some of these genes is accompanied by a duplication and transposition of the gene (the basic copy) to another region in the genome where it is transcribed. We present here physical maps of the basic and transposition-activated genes for two surface glycoproteins of Trypanosoma brucei, stock 427. In both cases the transposed segment starts 1-2 kb in front of the coding region and ends within the 3'-terminal region of the gene. The DNA segments flanking both transposed genes are indistinguishable and share a 6-kb stretch upstream and a 8-kb stretch downstream of the transposed segment not cut by several restriction endonucleases. The 5' borders of the two transposed segments are homologous and contain sequences present in many copies in the genome. A different repeated sequence has previously been found at the 3' edge of the transposed segment. The replicative transposition may, therefore, involve a unidirectional gene conversion initiated by base pairing between the edges of the transposed sequence and a single expression site elsewhere in the genome.

The effect of temperature and primer concentration on oligonucleotide-primed transcription has been studied using the separated strands of a well-defined natural DNA as template. Results were similar to those obtained in the homopolymer-directed model systems. At high temperature and excess primer concentration mainly primer-initiated RNA copies are synthesized. Omission of one ribonucleoside triphosphate also makes the termination specific. The unique RNA fragments thus obtained have been used to determine the perfectly-repeated sequence of 68 base pairs in this DNA.

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10-15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

Expression of surface antigen genes in Trypanosoma brucei occurs at expression sites located near telomeres. Since only one antigen is produced at a time, a mechanism must exist to prevent the simultaneous activity of multiple expression sites. Here we report that PstI and PvuII restriction sites in silent telomeric antigen genes are partially uncleavable , presumably as a consequence of DNA modification. The modification, which is absent in transcribed genes but returns after gene inactivation, may be specific for telomeric DNA because (1) it is not detected in non-telomeric genes; (2) modification is highest close to the telomere; (3) the level of modification in a telomeric gene is influenced by the size of the telomeric DNA segment downstream. Whether telomere modification is cause or consequence of antigen gene switch-off remains to be determined.

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.