A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.     

Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene

To examine whether the reduced shoot growth of abscisic acid (ABA)-deficient mutants of tomato is independent of effects on plant water balance, flacca and notabilis were grown under controlled-humidity conditions so that their leaf water potentials were equal to or higher than those of well-watered wild-type plants throughout development. Most parameters of shoot growth remained markedly impaired and root growth was also greatly reduced. Additional experiments with flacca showed that shoot growth substantially recovered when wild-type levels of ABA were restored by treatment with exogenous ABA, even though improvement in leaf water potential was prevented. The ability of applied ABA to increase growth was greatest for leaf expansion, which was restored by 75%. The ethylene evolution rate of growing leaves was doubled in flacca compared to the wild type and treatment with silver thiosulphate to inhibit ethylene action partially restored shoot growth. The results demonstrate that normal levels of endogenous ABA are required to maintain shoot development, particularly leaf expansion, in well-watered tomato plants, independently of effects on plant water balance. The impairment of shoot growth caused by ABA deficiency is at least partly attributable to ethylene.     

Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.     

Antibody-Secreting Cell Responses and Protective Immunity Assessed in Gnotobiotic Pigs Inoculated Orally or Intramuscularly with Inactivated Human Rotavirus

Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075–3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.