Yeast spt6-140 mutation, affecting chromatin and transcription, preferentially increases recombination in which Rad51p-mediated strand exchange is dispensable.

We have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57. Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat. The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively. Hyper-recombination levels are diminished in the absence of transcription. We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange. Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication. We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin. Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

Mitotic recombination in yeast: elements controlling its incidence.

Mitotic recombination is an important mechanism of DNA repair in eukaryotic cells. Given the redundancy of the eukaryotic genomes and the presence of repeated DNA sequences, recombination may also be an important source of genomic instability. Here we review the data, mainly from the budding yeast S. cerevisiae, that may help to understand the spontaneous origin of mitotic recombination and the different elements that may control its occurrence. We cover those observations suggesting a putative role of replication defects and DNA damage, including double-strand breaks, as sources of mitotic homologous recombination. An important part of the review is devoted to the experimental evidence suggesting that transcription and chromatin structure are important factors modulating the incidence of mitotic recombination. This is of great relevance in order to identify the causes and risk factors of genomic instability in eukaryotes.

Construction and genetic analysis of S. cerevisiae deletants of six novel ORFs from chromosome II.

We have constructed S. cerevisiae strains carrying genomic deletions of six ORFs from the left arm of chromosome II (YBL018c, YBL019w, YBL024w, YBL042c, YBL043w and YBL046w) in both FY1679 and W303 backgrounds. We have found that YBL018c is an essential gene in yeast, whereas the other five genes are non-essential. We have developed plasmids carrying deletion cassettes that can be used to delete any of the six genes in S. cerevisiae by transforming to G418-resistance, as well as centromeric plasmids containing the cognate genes.

Genetic stability and DNA rearrangements associated with a 2 x 1.1-Kb perfect palindrome in Escherichia coli.

We show that circular plasmids containing perfect palindromic regions of 2 x 1.1 kb can be propagated in sbcC strains of Escherichia coli, a result that is at variance with the well known observation that lambda DNA cannot tolerate palindromic regions larger than 2 x 265 bp. However, a significant fraction of these palindrome-containing plasmids can be recovered from E. coli strains either as linear molecules with hairpins at their ends or as head-to-head dimers, both in a RuvC-and RusA-independent manner. Our results suggests that large palindromes may form cruciforms in E. coli. However, palindrome-associated DNA rearrangements occur by a process that does not require any known cruciform resolvase activity. Our data support a replication-dependent model for the induction of DNA rearrangements by perfect palindromes.

Antimalaric effect of an alcoholic extract of Artemisia ludoviciana mexicana in a rodent malaria model.

Chloroquine resistance of Plasmodium falciparum first and of P. vivax more recently, stimulated the search for new antimalarics. Chinese investigators have introduced new compounds obtained from extracts of Artemisia annua which possess an antimalaric active principle different from those of the drugs in use. In Mexico eight species of Artemisia have been described and among them just A. ludoviciana has been empirically used in the treatment of intermittent fever. To know whether mexican Artemisia had antimalaric activity several in vivo experiments were performed. Different type of extracts from two Artemisia species were prepared and assayed in five different doses on mice infected by Plasmodium yoelii yoelii, in a four-day test scheme. Here, only the results of the assays on ethanolic extract of A. ludoviciana are presented. The results of the in vivo experiments showed that the parasite reproduction was inhibited up to 98.6% at the fifth day, as compared with the controls; the ED50 was of 29.2 mg/kg and the SM50 of 28.7. We looked after the presence of artemisinin in the ethanolic extract, without success.

Differential intrachromosomal hyper-recombination phenotype of spt4 and spt6 mutants of S. cerevisiae.

In order to test whether mutations affecting transcription also affect DNA recombination, we have determined the influence of a number of snf/swi and spt/sin transcriptional regulatory mutations on the recombination of a DNA inverted repeat. Among the nine different mutations analyzed, we found that spt4 and spt6 confer a significant hyper-recombination phenotype. Both mutations produced increases in the frequencies of reciprocal exchange/gene conversion and deletion events ranging from 1- to 15-fold above the wild-type levels, as determined in six direct repeat systems and one inverted repeat. The frequency of mitotic recombination between homologs, determined at one chromosomal locus, was not affected. We discuss the intrachromosomal hyper-recombination phenotype of spt4 an spt6 on the basis of the possible functions of SPT4 and SPT6 on transcriptional regulation and on chromatin structure.

Estimation of the time required by the malaria parasites to cross the digestive tract to reach blood in mice inoculated by the oral route.

In a previous report we described the transmission of the malaria parasites by the oral route in a murine model. Later, we performed some experiments to demonstrate the transmission of malaria infection by cannibalism. Now we commence to look for the site, mechanism and stages of the parasite involved in crossing the alimentary canal to reach blood and start the infection. To know the invasive stage of the parasite and the way it penetrates, we wanted first to find the level of the digestive tract through which the parasites cross, to restrict the area to be studied. We proposed that the crossing place would be known, if the crossing time of the parasite could be established. Mice were orally inoculated with Plasmodium yoelii yoelii infected blood and their blood was transferred at different times into clean recipient mice intraperitoneally. Malaria infection detected in recipient mice proved that infective forms of the parasite were circulating in the donor mice at the time the blood samples were taken. In this way, we observed that: i) although most parasites required between 2 to 10 min for crossing the alimentary canal, in some case the process can last for 22 hrs; ii) the parasites circulate in blood for variable periods of time (only two minutes in the shortest, and from 10 min on in the longest) being infective to blood recipients. Most orally-inoculated mice whose blood infects other mice, became transient carriers of parasites unable to establish in them.

Experimental transmission of Babesia microti infection by the oral route.

Previously we have described the transmission of malaria by the oral route in a murine model. Due to the similarities between Plasmodium and Babesia, we tried to reproduce oral transmission in parasites of the latter genus by ingestion of infected blood and by cannibalism. In the first case, experimental mice were inoculated orally with 20, 50, or 100 microliters of Babesia microti-infected blood, and in the second, each fasted experimental mouse was offered the corpse of an infected mouse serving as the bait inoculum. B. microti infection was acquired by 3.7% of all experimental animals orally inoculated with infected blood and by 15.1% of all mice inoculated by cannibalism. The approximate period of prepatency ran from 2 to 4 weeks. No control mouse acquired the infection. This represents the first time that oral transmission of babesiosis has been described. This kind of transmission may be present in nature. Babesiosis may be acquired and maintained in nature in the absence of ticks.

Experimental transmission of murine malaria by the oral route.

A total of 116 young male CD1 mice were orally inoculated with mouse blood; half of the animals received 0.2 ml of uninfected blood and the others were given 0.2 ml of Plasmodium berghei yoelii-infected blood in six experiments performed at different times. Almost 30% of the experimental mice acquired malaria as demonstrated by the observation of parasites in their blood. In no case were parasites found in the blood of control mice. Rodent malaria parasites may be transmitted to CD1 mice by the ingestion of mouse blood parasitized by P. b. yoelii. As far as we know, this study represents the first demonstration of oral transmission of murine malaria. Oral transmission studies in this mouse-Plasmodium model may produce very important information on the biology of the malaria parasites.

On the current existence of a coccidial line of development in the malaria parasites: a theory.

Most opinion favors the origin of the malaria parasites from a coccidial ancestor. It is assumed that whatever the process through which the coccidia differentiated into a Plasmodium this phenomenon very probably occurred millions of year ago, and during that differentiation process the original coccidia vanished. Therefore it has never been repeated. At the light of some experiments the existence, at the present time, of a coccidial cycle of development in the malaria parasites, is proposed. The connection routes and mechanisms through which the malaria parasite changes to a coccidial life, and the routes in reverse are exposed. Transmission of the malaria-coccidial forms is suggested.

[Standardization of the histamine liberation test]. / Estandarización del test de liberación de histamina.

The application of the histamine liberation test to the diagnosis and follow-up of allergic processes has been pursued by many authors. Since Shore first described a manual fluorimetric technique, efforts have been directed at automating the system and reducing the sample volume. According to the latest contributions by Moneo et al., from 300 to 400 determinations can be carried out in a single work day, and from 40 to 50 determinations with 10 ml of blood. In the present work we present our experienced in updating the technique and the system of preparation of a histamine standard, which we consider of great practical use in the application of this technique routine. Using the standard solution as reference, we calculated total and basal histamine levels in 342 presumed healthy individuals; the mean values were 69.5 ng/ml for total histamine and 8% for basal histamine in males, and 71 ng/ml for total histamine and 6% for basal histamine in females. Age range was from 17 to 57 years, and there were no differences between sexes. The maximum value was 176 ng/ml and the minimum 32 ng/ml. The reaction of O-phthaldialdehyde with histamine is not chemically specific. In the presence of light, aldehydes self-polymerize; moreover, O-phthaldialdehyde can react with other products in addition to histamine. The first problem is avoided by protecting the reagents from light, and the second by using specific absorption and emission filters which do not pick up signals from reaction side products. Nevertheless, at the quantitative level these effects are negligible when compared to other causes which, although not part of the chemical reaction, are also involved in the process (antigen standardization, etc). By means of this technique, from 300 to 400 determinations can be carried out in a single work day, making it possible to use this technique in the daily work routine.

Quantification of antibodies against factor VIII by an immunonephelometric method in a continuous-flow system.

A new immunonephelometric method is described for the quantification of antibodies against factor VIII in sera from patients suffering from haemophilia A. We have been able to reduce the reaction time to only 16 minutes, which permits its application to emergency cases. Applying Spearman's test, we have correlated the results obtained by the immunonephelometric and Ruggieri's methods, obtaining r = 0.87, P < 0.01 for n = 11. From the reproducibility studies we obtained SD = 1.16 as maximum dispersion value. Because it is a fully automated method in a continuous-flow system, the possible manual errors are reduced to a minimum; 60 determinations can be carried out in 1 hour.