Unusual sequence element found at the end of an amplicon.

In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 X d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed.

Interference footprinting analysis of telomerase elongation complexes.

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.

DNA molecules can drive the assembly of other DNA molecules into specific four-stranded structures.

Single-stranded DNA molecules containing clustered G-repeats can be assembled into various four-stranded structures linked by G-quartets. Here, we report that such molecules can also drive the assembly of other DNA molecules containing G-repeats into specific four-stranded structures. In these assays, the oligonucleotides 5'-CAGGCTGAGCAGGTACGGGGGAGCTGGGGTAGATTGGAATGTAG-3' (oligo D) and 5'-CGGGGGAGCTGGGGT-3' (oligo B), consisting of sequences found in immunoglobulin switch regions, were annealed in a buffer containing K+ and the annealing products were analyzed by polyacrylamide gel electrophoresis. This analysis revealed that whereas annealing of each oligo alone produced four-stranded structures designated D2 and B2, annealing of mixtures containing both oligos produced additional complexes designated D2* and B2*. D2* and B2* were found to contain only D molecules and only B molecules, respectively. The yield of D2* increased and the yield of B2* decreased, as the concentration ratio oligo B/oligo D was increased. These results indicated that B can drive the assembly of D into D2* and D can drive the assembly of B into B2*. Further studies revealed that while the assembly of D2 followed a second order kinetics, the B-driven assembly of D2* followed a first order kinetics. Dimethyl sulfate footprinting indicated that both D2 and D2* are four-stranded structures containing two parallel and two antiparallel chains. In addition, annealing of D mixed with various B mutants showed that only mutants containing two G-clusters can drive the assembly of D2*. Based on these data, we propose that in the process of D2* assembly, a four-stranded intermediate containing B and D is formed and then dissociates into D2* and B in a rate-limiting first order reaction. Driver mechanisms of this type may cause formation of specific four-stranded structures at G-rich chromosomal sites, thereby regulating processes such as recombination and telomere synthesis.

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin.

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.

'Illegitimate' recombination events in polyoma-transformed rat cells.

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.

Solitary eosinophilic granuloma of sternum: case report with review of the literature.

A solitary lesion in the distal sternum in a 30-year-old woman caused by eosinophilic granuloma (EG) is reported. Bone scan with 99Tcm was negative. Laboratory tests were completely normal. Although EG bone lesions have been discussed extensively in the literature only one similar case of solitary EG of the sternum has been published to date. A comprehensive summary of the literature is presented.

Use of percutaneous nephrostomy in hydronephrosis of pregnancy.

Traditional methods of urinary diversion in pregnancy include retrograde passage of ureteral catheter or stents and operative nephrostomy. These techniques are, however, associated with the use of anesthesia, are technically difficult to perform, and may induce labor. We report the use of percutaneous nephrostomy in four pregnant patients, two with obstruction due to ureteral calculi and two with infected hydronephrosis. The procedure provided rapid relief from pain and pyosepsis, and allowed uneventful continuation of the pregnancy to full-term, with preservation of renal function.

[Spontaneous rupture of a hepatocellular carcinoma successfully managed by embolization of the hepatic artery].

An 87-year old woman was admitted with a clinical picture of hypovolemic shock, preceded by a few weeks of abdominal pain. On admission, blood urea was high, the hemoglobin 5 g% and the CT scan revealed a large mass in the right hepatic lobe and free fluid in the abdominal cavity. A tentative diagnosis of ruptured hepatoma with spontaneous intraperitoneal bleeding was made. The diagnosis was confirmed by angiography and selective embolization of the hepatic artery stopped the bleeding. The patient left hospital a few days later.

[Gallbladder polyps].

There is a relatively high incidence of asymptomatic gallbladder polyps in the general population (1.5-9.5%). We present a 61-year-old woman who had a 22 x 37 mm polyp of the gallbladder.

[Transrectal ultrasound-controlled needle aspiration of a prostatic cyst].

Prostatic cysts are common and are usually acquired and asymptomatic. Congenital prostatic cysts, which may originate from the wolffian system or from remnants of the Muellerian duct, are rare and may cause obstructive symptoms in young adults. Due to the availability of transrectal ultrasonography an increased number of cases of prostatic cysts are being diagnosed. We report a 36-year-old man with a congenital prostatic cyst which caused increasing pain during ejaculation and decreased the force of the urinary stream. It was diagnosed by ultrasonography and treated successfully by ultrasound-controlled, transrectal needle aspiration. There was immediate, complete disappearance of symptoms and no complications.

[Epidermoid cyst of the spleen].

Splenic cysts are rarely found or diagnosed. Excluding cases of trauma, the events preceding their development have not been fully understood. We describe a 22-year-old woman in her 34th week of pregnancy in whom ultrasound revealed a cystic lesion 8 x 10 cm. in diameter in the left upper abdomen. Further imaging tests followed by laparotomy confirmed the splenic origin of the cyst. Splenectomy was performed and the lesion was histopathologically defined as an epidermoid cyst.