Disruption of the expression of a non-coding RNA significantly impairs cellular differentiation in Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing "tachyzoites" and latent "bradyzoite" tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. We have developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites. One of these mutants has an insertion disrupting a locus that encodes a developmentally regulated non-coding RNA transcript, named Tg-ncRNA-1. Microarray hybridizations suggest that Tg-ncRNA-1 is involved in the early steps of bradyzoite differentiation. Since Tg-ncRNA-1 does not contain an open reading frame, we used the algorithm Coding Potential Calculator (CPC) that evaluates the protein-coding potential of a transcript, to classify Tg-ncRNA-1. The CPC results strongly indicate that Tg-ncRNA-1 is a non-coding RNA (ncRNA). Interestingly, a previously generated mutant also contains an insertion in Tg-ncRNA-1. We show that both mutants have a decreased ability to form bradyzoites, and complementation of both mutants with wild-type Tg-ncRNA-1 restores the ability of the parasites to differentiate. It has been shown that an important part of bradyzoite differentiation is transcriptionally controlled, but this is the first time that a non-coding RNA is implicated in this process.

Minimum entropy decomposition: unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences.

Molecular microbial ecology investigations often employ large marker gene datasets, for example, ribosomal RNAs, to represent the occurrence of single-cell genomes in microbial communities. Massively parallel DNA sequencing technologies enable extensive surveys of marker gene libraries that sometimes include nearly identical sequences. Computational approaches that rely on pairwise sequence alignments for similarity assessment and de novo clustering with de facto similarity thresholds to partition high-throughput sequencing datasets constrain fine-scale resolution descriptions of microbial communities. Minimum Entropy Decomposition (MED) provides a computationally efficient means to partition marker gene datasets into 'MED nodes', which represent homogeneous operational taxonomic units. By employing Shannon entropy, MED uses only the information-rich nucleotide positions across reads and iteratively partitions large datasets while omitting stochastic variation. When applied to analyses of microbiomes from two deep-sea cryptic sponges Hexadella dedritifera and Hexadella cf. dedritifera, MED resolved a key Gammaproteobacteria cluster into multiple MED nodes that are specific to different sponges, and revealed that these closely related sympatric sponge species maintain distinct microbial communities. MED analysis of a previously published human oral microbiome dataset also revealed that taxa separated by less than 1% sequence variation distributed to distinct niches in the oral cavity. The information theory-guided decomposition process behind the MED algorithm enables sensitive discrimination of closely related organisms in marker gene amplicon datasets without relying on extensive computational heuristics and user supervision.

Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state.

Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states--genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.

Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies.

We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.

Human progesterone receptor polymorphisms and implantation failure during in vitro fertilization.

OBJECTIVE: Polymorphic variants of the human progesterone receptor gene have been described, but their potential influence on in vitro fertilization outcome have not been studied. STUDY DESIGN: DNA was available from 317 women (cases) who had >or=2 embryo transfers without a clinical pregnancy and from 288 women (control subjects) who became pregnant after 1 in vitro fertilization attempt. With protocols that were specific for H770H (C/T genotype) and +331G/A polymorphism, amplification of polymorphism fragments, digestion with restriction enzymes, and gel visualization were performed. RESULTS: Allele frequencies for the 2 variants were 15.5% for H770H T and 4.1% for +331A in cases and 17.9% and 3.6% in control subjects, respectively. Both polymorphisms were rare among non-white subjects. Possession of a H770H T allele was associated with a decreased risk for implantation failure, which was nonsignificant overall but significant in women aged <35 years (adjusted relative risk, 0.52 [95% CI, 0.28, 0.97]). Possession of a +331A allele was associated with an increased risk of implantation failure, which was nonsignificant overall but significant in women who weighed <135 pounds (adjusted relative risk, 3.85 [95% CI, 1.20, 12.30]). The +331G/A polymorphism tended to increase the risk for implantation failure in women with an H770H C/C genotype, but not in women with a C/T or TT genotype (P=.09). Among white women who were H770H C/C, there was a significant trend (P=.03) in the proportion of +331G/A carriers as the number of implantation failures increased, from 8.6% of women with 0 failed attempts to 40% among women with >or=5 failed attempts. CONCLUSION: Although human progesterone receptor polymorphisms do not clearly affect the risk for implantation failure in most women who undergo in vitro fertilization, the likelihood that a woman carries the +331G/A human progesterone receptor polymorphism increases with the number of failed attempts at implantation.

A functional polymorphism in the promoter of the progesterone receptor gene associated with endometrial cancer risk.

Excessive estrogen stimulation unopposed by progesterone strongly predisposes to endometrial cancer. Because the antiproliferative effect of progesterone requires the progesterone receptor (PR), which exists in two isoforms, PR-A and -B, we reasoned that variants in the PR gene may predispose to endometrial cancer. We found six variable sites, including four polymorphisms in the hPR gene and five common haplotypes. One promoter region polymorphism, +331G/A, creates a unique transcription start site. Biochemical assays showed that the +331G/A polymorphism increases transcription of the PR gene, favoring production of hPR-B in an endometrial cancer cell line. Using a case-control study nested within the Nurses' Health Study cohort, we observed a statistically significant association between the +331G/A polymorphism and the risk of endometrial cancer, which was even greater in overweight women carriers. After including a second population of controls, these associations remained intact. Our findings suggest that the +331G/A hPR gene polymorphism may contribute to endometrial cancer risk by increasing expression of the hPR-B isoform.