On the role of repeated sequences 5' to variant surface glycoprotein genes in African trypanosomes

In African trypanosomes, the DNA region situated upstream from all active and some silent variant surface glycoprotein genes (VSG genes) has a repetitive structure. This region is composed of a variable number of tandem repeats of an A + T-rich sequence which lacks the recognition sites for most commonly used restriction endonucleases, and is thus called 'barren region'. The length of the barren regions varies in different trypanosome variants from 0.2 to many kb. We have characterized the barren region upstream from the active VSG gene in two independent Trypanosoma equiperdum variants expressing the same VSG gene in the same expression site. To analyse the junction point between the expression site and the inserted gene, these two barren regions were cloned and sequenced. The longer barren region contains 14 repeats and the other contains two repeats. In both cases the junction point has been shown to lie within a repeat but different repeats were used in each case. These results argue that the repeats are important for the insertion of the duplicated-transposed gene into the expression site and that any repeat can be used.     

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.     

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.     

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).     

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.     

Highly sensitive detection of cytotoxicity using a modified HSP70B' promoter

We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B' gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B' promoter. 5'- and 3'-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B' gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B' gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B' promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B' promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B' promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.     

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.     

Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells.

Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system. It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site. Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene. beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons. Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides. Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.     

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Interaction of hnRNP A2/B1 isoforms with telomeric ssDNA and the in vitro function

Overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, especially of B1 has been reported as a useful marker to detect cancers in early stage, although the biological reason is not clear. A2/B1 proteins were previously reported to bind telomeric DNA repeats. Alternative splicing of A2/B1 gene produces abundant A2, less abundant B1, and testis-specific minor isoforms B0a and B0b. In this study, B1 and B0b that have the N-terminal 12 amino acid insertion were suggested to have higher affinities to telomeric single-stranded DNA (ssDNA) than A2 and B0a. Kinetic analyses using purified B1 and B0b indicated that they interact dynamically with a single array of telomeric repeats. Furthermore, functional assays demonstrated that B1 and B0b bind with telomeric repeats in a tandem fashion and protect them from a nuclease and promote telomerase activity. A2/B1 proteins, especially B1 and B0b, may function as telomeric ssDNA-binding proteins in cancer and reproductive cells.     

Breakage--reunion and copy choice mechanisms of recombination between short homologous sequences.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.