The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodified base J in both strands.

We have previously shown that nuclear DNA of bloodstream from Trypanosoma brucei contains a novel base beta-glucosyl-hydroxymethyluracil, called J. Base J is enriched in minichromosome fractions but not in the minichromosome internal repeats, suggesting the association of J with telomeric DNA. To test whether J is present in the long telomeric (GGGTTA)n repeat arrays, which are 2-26 kb in T.brucei, we have purified these arrays both by hybrid selection and by isolating 2-26 kb fragments from DNA digested with multiple restriction enzymes. We find that in purified telomeric repeats approximately 13% of T is replaced by J, compared to 0.8% in total DNA, and we estimate that approximately 50% of the total J is in these repeats. Highly purified complementary strands of the repeats were obtained by alkaline CsCl equilibrium centrifugation. In the (TAACCC)n strand 14% of T was replaced by J. In the (GGGTTA)n strand approximately 36% of the second T was replaced by J; the first T was not detectably replaced. Modified bases have not been found in telomeric repeats before. How the bulky base J affects telomere function and structure in bloodstream form trypanosomes remains to be determined.     

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.     

Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA.

Integrated retroviral DNA is flanked by short direct repeats of the target DNA. The length of these repeats is specific for the provirus that is integrated (H.E. Varmus, in J.A. Shapiro, ed., Mobile Genetic Elements, 1983). For the human immunodeficiency virus type I (HIV-1), the length of the direct repeats in the target DNA was shown to be 5 bp in one case (Muesing et al., Nature [London] 313:450-458, 1985) and 7 bp in another (Starcich et al., Science 227:538-540, 1985). One possible explanation for this discrepancy is that the direct repeats flanking HIV-1 proviruses are variable. To investigate this, we analyzed the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. In each clone the provirus was flanked by a 5-bp direct repeat of human DNA. Analysis of the proviral clone previously described as being flanked by a 7-bp direct repeat of target DNA (Starcich et al., op. cit.) revealed that this clone was flanked by a 5-bp repeat instead. Therefore, we conclude that HIV-1 proviruses are flanked by 5-bp direct repeats of human DNA. The sequences of the 5-bp duplications from the different proviral clones do not have any apparent similarity to each other or to HIV-1 DNA.     

Two-dimensional graphic analysis of DNA sequence homologies.

We describe a computer program designed to facilitate the pattern matching analysis of homologies between DNA sequences. It takes advantage of a two-dimensional plot in order to simplify the evaluation of significant structures inherited in the sequences. The program can be divided into three parts, i) algorithm for search of homologies, ii) two-dimensional graphic display of the result, iii) further graphic treatment to enhance significant structures. The power of the graphic display is presented by the following application of the program. We conducted a search for direct repeats in the mouse immunoglobulin kappa-chain genes. Both the five J DNA sequences and other shorter repeats were found. We also found a longer stretch of homology that could indicate the presence of duplicated DNA in the J4, J5 region.     

Identical 3250-bp deletion between two AluI repeats in the ADA genes of unrelated ADA-SCID patients.

Recently, we investigated a Belgian patient with severe combined immune deficiency caused by a dysfunction of the gene for adenosine deaminase (ADA-SCID), which was found to be due to a 3.2-kb deletion spanning the promoter and the first exon of the ADA gene (Berkvens et al., 1987, Eur. J. Pediatr. 146:329). No ADA-specific RNA could be detected in primary fibroblasts derived from this patient. In the present paper we establish via direct sequencing of in vitro amplified DNA that the 3250-bp deletion is due to a recombination within the left arms of two direct AluI repeats. This mutation is identical to one reported for an unrelated patient in the United States (Markert et al., 1988, J. Clin. Invest. 81:1323-1327).     

Linkage map of the fragments of herpesvirus papio DNA.

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978).     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.     

Expression of the human DNA glycosylase hSMUG1 in Trypanosoma brucei causes DNA damage and interferes with J biosynthesis

In kinetoplastid flagellates such as Trypanosoma brucei, a small percentage of the thymine residues in the nuclear DNA is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J), mostly in repetitive sequences like the telomeric GGGTTA repeats. In addition, traces of 5-hydroxymethyluracil (HOMeUra) are present. Previous work has suggested that J is synthesised in two steps via HOMedU as an intermediate, but as J synthesising enzymes have not yet been identified, the biosynthetic pathway remains unclear. To test a model in which HOMeUra functions as a precursor of J, we introduced an inducible gene for the human DNA glycosylase hSMUG1 into bloodstream form T.brucei. In higher eukaryotes SMUG1 excises HOMeUra as part of the base excision repair system. We show that expression of the gene in T.brucei leads to massive DNA damage in J-modified sequences and results in cell cycle arrest and, eventually, death. hSMUG1 also reduces the J content of the trypanosome DNA. This work supports the idea that HOMeUra is a precursor of J, freely accessible to a DNA glycosylase.     

Simple human DNA-repeats associated with genomic hypervariability, flanking the genomic retroposons and similar to retroviral sites.

Earlier we found a human hypervariable genomic region (GVR). The DNA hybridization probe isolated from this region detects multiple hypervariability of restriction DNA fragments from genomic loci. The sequencing data suggest that the genomic instability and variability are associated with tandem DNA repeats. The DNA hybridization probe contains two families of simple DNA repeats designated as 'apo' and 'tau'. The (TC)n-rich family of DNA 'tau'-repeats bears some similarity to the simple transcribed repeats of Drosophila virilis, simple repetitive motifs of the human proenkephaline gene exon 1, and short sites of retroviral LTR ends. Apo-repeats show an unusual similarity to Rauscher viral env gene site. Besides GVR, apo- and tau-like repeats are localized in other genomic loci and can form separate tandem clusters and terminal repeats flanking certain copies of retroposons (Alu-SINES).     

Eucaryotic transposable genetic elements with inverted terminal repeats

DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.     

Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus.

The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.     

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.     

Short direct repeats mediate spontaneous high-frequency deletions in DNA of minute virus of mice.

Previous work (E. A. Faust and D. C. Ward, J. Virol. 32:276-292, 1979) revealed a remarkably high rate of spontaneous deletion in viral DNA during lytic infection of cultured murine cells with minute virus of mice (MVM), an autonomous parvovirus. In the present study, we have isolated plasmid and phage recombinants containing MVM DNA inserts bearing deletions and we have determined the DNA sequence spanning three deletion junctions. The deletions, which average 3 kilobases in length, occur between pairs of perfectly homologous 4- to 10-base-pair direct repeats, such that one copy of the repeated sequence is lost, whereas the other remains behind at the deletion junction. When compared, the three sets of direct repeats exhibit no apparent sequence homology and have an A + T content of between 50 and 80%. These results indicate that 4- to 10-base-pair homologies mediate spontaneous deletion formation in the MVM genome and highlight parvoviruses as novel model systems for studies of this ubiquitous pathway of genetic variation.     

Timing and sequence requirements defined for embryonic maintenance of imprinted DNA methylation at Rasgrf1

Epigenetic programming is critical for normal development of mammalian embryos. Errors cause misexpression of genes and aberrant development (E. Li, C. Beard, and R. Jaenisch, Nature 366:362-365, 1993). Imprinted genes are important targets of epigenetic regulation, but little is known about how the epigenetic patterns are established in the parental germ lines and maintained in the embryo. Paternal allele-specific expression at the imprinted Rasgrf1 locus in mice is controlled by paternal allele-specific methylation at a differentially methylated domain (DMD). DMD methylation is in turn controlled by a direct repeat sequence immediately downstream of the DMD which is required for establishing Rasgrf1 methylation in the male germ line (B. J. Yoon et al., Nat. Genet. 30:92-96, 2002). To determine if these repeats have a role in methylation maintenance, we developed a conditional deletion of the repeat sequence in mice and showed that the repeats are also required during a narrow interval to maintain paternal methylation of Rasgrf1 in developing embryos. Removing the repeats upon fertilization caused a total loss of methylation by the morula stage, but by the epiblast stage, the repeats were completely dispensable for methylation maintenance. This developmental interval coincides with genome-wide demethylation and remethylation in mice which most imprinted genes resist. Our data show that the Rasgrf1 repeats serve at least two functions: first, to establish Rasgrf1 DNA methylation in the male germ line, and second, to resist global demethylation in the preimplantation embryo.     

Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells

Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.     

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).