Identification of Berenil Target Sites in Plasmid pBR322.

Berenil, a minor groove DNA binding molecule, has been extensively used in veterinary medicine. Modeling studies have suggested that berenil binds to A/T rich regions on the DNA and the product of this interaction causes the formation of crosslinks between opposite DNA strands. These crosslinks could potentially inhibit fundamental biological processes including transcription and DNA replication. We had previously used the pBR322 genome as a model system to investigate the role of A/T sequences on berenil activity. We reported that the insertion of poly(dA)poly(dT) sequences into the pBR322 genome causes replication inhibition of the recombinant plasmids when cultures were exposed to berenil. However, we noticed that even in the absence of these sequences the parental plasmid replication was also inhibited, albeit less than the recombinants. This observation led us to the present study were we attempted to identify the location of natural berenil target sites in the pBR322 genome. Through a combination of deletion analysis, recombinant DNA and a replication assay we uncovered a 378 bp DNA fragment that has all the hallmarks of a berenil target site. A recombinant plasmid lacking this region is more refractive to the drug than the parental plasmid, and another variant containing and extra copy of this region increases the susceptibility of the plasmid towards berenil. The 378 bp region is about 60% A/T rich and contains about 21 potential berenil binding sites.

High expression of plasmid-encoded tetracycline resistance gene in E. coli causes a decrease in membrane-bound ATPase activity.

Expression of the plasmid pBR322-encoded tetracycline-resistant gene (tet) is known to cause other pleiotropic effects in addition to mediating the efflux of tetracycline from bacterial host cells. We have recently reported that expression of the tet gene in plasmid pKH47, a high-copy-number derivative of pBR322, causes growth inhibition of Escherichia coli cells harboring this plasmid. In this paper we report that reduced membrane-bound ATPase activity is found in E. coli cells containing plasmid pKH47. This effect is dependent on the presence of an intact tet gene and reduces the ability of the cells to grow in a minimum medium containing succinate as the sole carbon source. The same effect is more dramatically observed in cells containing an unrelated plasmid in which tet gene expression is under the control of the tac promoter.

E. coli growth inhibition by a high copy number derivative of plasmid pBR322.

We have observed that plasmid pKH47, a pBR322-derivative containing a 100bp poly(dA)-poly(dT) insertion, causes growth inhibition of host E. coli cells harboring it. In this paper we show that this inhibitory effect is due to an increased copy number property of this plasmid, which is turn leads to an over expression of the plasmid-encoded tet gene. Our work also indicates that contrary to other pleiotropic effects caused by the tet gene product, which solely depend on the expression of the 5' end of the gene, growth inhibition requires an intact tet gene. In addition we present the isolation of an E. coli mutant that is refractive to the inhibitory effect of pKH47 and shares some properties with the parental bacteria containing plasmid pKH4.

Kinetoplast DNA minicircle binding proteins in a Leishmania Spp: interference of protein DNA interaction by berenil.

A kinetoplast DNA minicircle of a Leishmania Spp. binds to several proteins of the kinetoplast Lysates of kinetoplasts of Leishmania grown in the presence of berenil show complete disappearance of some of these protein bands, while the rest of the proteins present appear as much less intense bands in South Western blots when probed with either the conserved or variable regions of the minicircle or whole minicircle DNA. The conserved region of minicircle DNA complexed with berenil in vitro also fails to interact with the DNA binding proteins of kinetoplast of untreated cell in South Western blots. Since berenil induces dyskinetoplasty of kinetoplastidae, the results indicate that interference of protein-DNA interaction in the presence of berenil may be the primary event in making organisms dyskinetoplastic.

Unintended pregnancy and sex education in Chile: a behavioural model.

This study analysed factors associated with unintended pregnancy among adolescent and young adult women in Santiago, Chile. Three variations of a behavioural model were developed. Logistic regression showed that the effect of sex education on unintended pregnancy works through the use of contraception. Other significant effects were found for variables reflecting socioeconomic status and a woman's acceptance of her sexuality. The results also suggested that labelling affects measurement of 'unintended' pregnancy.

PIP: Using data from the Santiago Young Adult Reproductive Health Survey on 306 females aged 15-24 years living in greater Santiago in 1988 and having experienced premarital intercourse, the authors model the relationship of sex education and unintended pregnancy using social learning theory which posits that any health behavior is part of a dynamic model in which an individual's behavior is determined by the interaction of personal factors and environmental influences. Three variations of a behavioral model are developed. They found that women who had sex education before first intercourse and used contraception at that intercourse were one-third as likely to have a later unintended pregnancy as those with sex education who did not use contraception at first intercourse. Women who did not have sex education and who did not use contraception at first intercourse, however, were less likely to have an unintended pregnancy than women with no sex education who did use contraception. Across all models, moral ambivalence about premarital sexual activity is related to having an unintended pregnancy, Catholic women are twice as likely to have an unintended pregnancy as non-Catholics, and women from poorer backgrounds are less likely to use contraception and more likely to have early pregnancies. How women define their pregnancies can also affect the measurement of unintended pregnancy. It is important to effectively teach the use of contraceptives to reduce unintended pregnancies among adolescents and young adults who have premarital sexual intercourse.

Adhesion to and invasion of cultured human cells by Bartonella bacilliformis.

Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers. The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells. Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells. There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae. Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles. These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis.

Premarital sexual activity and contraceptive use in Santiago, Chile.

The Santiago Young Adult Reproductive Health Survey was conducted in 1988 to examine the sexual behavior of and contraceptive use among young adults in Chile. The survey was based on multistage household probability samples of 865 women and 800 men aged 15-24 who were living in Santiago in 1988. Findings show that 35 percent of females and 65 percent of males had had premarital intercourse. Among those who had done so, the median age at first experience was 18.4 years for women and 16.4 years for men. Only 20 percent of females and 19 percent of males used contraceptives at first premarital intercourse. Use of contraceptives increased with age at the time of that event. Fertility data reveal that 70 percent of first births were premaritally conceived, and more than one-third of these were born prior to union. The high rates of premarital and unintended pregnancy among young women and the low prevalence of effective contraceptive use indicate a need for greater emphasis on sex education and family planning services directed at adolescents and unmarried young adults in Santiago.

[Reproductive health survey of young adults in greater Santiago]. / Encuesta de salud reproductiva en adultos jóvenes del Gran Santiago.

In 1988 a survey was carried out in order to obtain information on knowledge about reproduction, sexual activity, attitudes, and use of contraceptive methods among residents between 15 and 24 years of age in Greater Santiago. For this purpose, a multistage, self-weighted, non-replacement probability sample was chosen from the entire Santiago urban area. After 2,898 households were visited, 865 women and 800 men were selected and interviewed. For the interview, a questionnaire with 156 questions was developed; many questions were similar to those included in similar surveys in Brazil and Guatemala. The interviewers were professionals who had received prior training. Although 75% of the interviewees had attended sex education classes, they had erroneous ideas on various basic subjects. Sixty-nine percent of the women interviewed had undergone menarche before attending these classes. In addition, 35.4% of the women and 65.0% of the men had had sexual relations prior to marriage, and less than 20% had used any contraceptive method. More than 60% of the interviewees who had children had conceived them before marrying. These findings point up the necessity of offering sex education classes for children and young people, as well as facilitating their access to family planning services, in order to decrease the number of illegitimate and unwanted children that are born in Chile.

Survey of reproductive health in young adults, greater Santiago, 1988.

This article reports the results of an interview survey with 1,665 residents of Metropolitan Santiago 15-24 years old. The survey, which dealt with various aspects of reproductive health, indicated that the sex education received by 75% of the subjects generally failed to convey an accurate knowledge of the basic concepts of sexuality; that use of contraceptive methods was very limited; that approximately 25% of the 865 women interviewed had been pregnant at some time; and that 40% of all the pregnancies were unplanned. These findings demonstrate a need to begin effective sex education programs and to provide adolescent services commensurate with the circumstances of modern life.

Catenated dimers and knotted DNA structures: putative intermediates in the replication of T. cruzi kinetoplast minicircle DNA.

Upon centrifugation of gently lysed T. cruzi cells through a sucrose gradient, a free DNA fraction was shown to contain catenated dimers and knotted DNA structures. Southern hybridization and electron microscopic studies indicated that both of these structures derived from minicircle DNA, the major component of T. cruzi kinetoplast DNA. Partial denaturation analysis of a random population of catenated dimers suggests that these structures may have arisen from a late stage in the replication of minicircle DNA. On the other hand, the T. cruzi knotted minicircles we have isolated appear to be very similar to trefoil structures recently reported and implicated as replicative intermediates in two other trypanosoma species.

[Reproductive health survey in young adults: sexual experience and use of contraceptives in Santiago, Chile]. / Encuesta de salud reproductiva en adultos jóvenes: experiencia sexual y uso de anticonceptivos en Santiago, Chile.

A self weighted, 2 stage probability sample of households in greater Santiago was obtained to select women and men between 15 and 24 years of age in order to obtain information about knowledge of sexual aspects, sexual activity and use of contraception. 35% of females and 65% of males declared to have had premarital sexual intercourse. Mean age at first premarital coitus was 18 and 16 years for women and men, respectively. Partners were 3.5 years older for females and 2.5 years older for males. A steady boy friend was the most common first sexual partner for females. Only 20% of women and 19% of males used contraception at first coitus. Rhythm, pills and withdrawal were the most commonly used methods. Less than 4 coituses in the 2 months preceding the survey were declared by 75% of subjects. All women declared themselves monogamous; 8% of males acknowledged more than one sexual partner in the last month.

An autonomously replicating sequence from HeLa DNA shows a similar organization to the yeast ARS1 element.

A HeLa DNA fragment, which may function as an anchorage point to the nuclear matrix for human chromosomes 1 and 2, also functions as an autonomously replicating sequence (ARS) in the yeast Saccharomyces cerevisiae. In the present report we show that this DNA fragment contains both bent DNA and an A-T rich region which appear to be associated with the ARS function. More interestingly, DNA sequence analysis shows that the spatial distribution of these features is strikingly similar to that found in the yeast ARS1 element.

Bidirectional sequencing of supercoiled plasmid DNA.

In this paper we show that restriction DNA fragments can prime DNA synthesis of a homologous supercoiled plasmid DNA. Using the dideoxyribonucleotide chain terminator method, newly synthesized truncated chains can be detached from the primers by restriction enzyme digestion. Therefore, by choosing DNA fragments flanked by two different restriction enzymes sites, nucleotide sequence information can be simultaneously obtained on both regions of the DNA surrounding the restriction fragment. The advantage of this sequencing approach over current methods is that no prior knowledge of the primary sequence is needed to find the nucleotide sequence of a given DNA fragment. Thus, synthetic primers are not required and internal sequences of a given clone can be easily accessed without the need of fragmenting the original construct. The method has been used with rapid plasmid preparations, thus considerable time and effort can be saved in the gathering of nucleotide sequence information.

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence.

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.

A unique deletion in pBR322 DNA caused by insertion of poly(dA).poly(dT).

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA).poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA).poly(dT) fragment of 10-20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.

Enriched autonomously replicating sequences in a nuclear matrix-DNA complex isolated from synchronized HeLa cells.

Nucleoids isolated from either synchronized or exponentially growing HeLa cells were digested with restriction enzymes to separate a nuclear matrix-bound DNA component from the rest. Partial libraries were constructed by inserting DNA fragments from both components into a yeast-bacteria plasmid vector. A random sample from these libraries was tested for ARS activity by a standard yeast transformation assay. We found that synchronization for DNA replication results in an enrichment for autonomously replicating sequences in the library constructed with the DNA component bound to the nuclear matrix.