The accurate description of a microbial community is an important first step in understanding the roles of its components in ecosystem function. A method for surveying microbial communities termed serial analysis of rRNA genes (SARD) is described here. Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA genes from microbial communities. These tags are ligated to form concatemers comprised of 20 to 40 tags which are cloned and identified by DNA sequencing. Four agricultural soil samples were profiled with SARD to assess the method's utility. A total of 37,008 SARD tags comprising 3,127 unique sequences were identified. A comparison of duplicate profiles from one soil genomic DNA preparation revealed that the method was highly reproducible. The large numbers of singleton tags, together with nonparametric richness estimates, indicated that a significant amount of sequence tag diversity remained undetected with this level of sampling. The abundance classes of the observed tags were scale-free and conformed to a power law distribution. Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community. Over 99% of the unique tags individually made up less than 1% of the community. Therefore, from either a numerical or diversity standpoint, taxa with low abundance comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function. SARD may provide a means to explore the ecological roles of these rare members of microbial communities in qualitative and quantitative terms.

Cystathionine-b-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.T HE first complete human genome sequence seeded the defining challenge of human genetics for the foreseeable future: interpreting the impact of variations in the sequences of individual human genomes. Comparative genome sequencing reveals an average of one single-nucleotide change per 1200 bp between any two individuals. In the absence of strong Mendelian inheritance and linkage, confirming that any human genotype actually caused a phenotype is a significant challenge given the approximately 3 million genetic variants per person. Indeed, 4000 traits of medical interest show evidence for inheritance but lack a clear determinant (Online Mendelian Inheritance in Man 2012). Next-generation sequencing within small pedigrees (Ng et al. 2010a,b;Fan et al. 2011), or a more narrowly defined clinical phenotype (Schubert et al. 1997), can sometimes disentangle the underlying contribution of a gene to disease. In this work we have taken an approach that complements both increased sequencing capacity and expanded phenotypic description. We used surrogate genetics to assay directly the function of allelic variants and then evaluate their potential contribution to phenotypes of clinical importance.Homocystinuria, elevated levels of the sulfur-containing metabolite homocystine in the urine, illustrates several Reference numbers for publicly available data; GenBank: L14577.1 (CBS); dbSNP: rs17849313 (A69P), rs2229413 (P70L), rs11700812 (R369P); SGD: YGR155W (CYS4) and YDR232W (HEM1 challenges inherent to elucidating the molecular bases of human geneti...

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.

Sterols and all nonsterol isoprenoids are derived from the highly conserved mevalonate pathway. In animal cells, this pathway is regulated in part at the transcriptional level through the action of sterol response element-binding proteins acting at specific DNA sequences near promoters. Here we extend at least part of this regulatory paradigm to the ERG10 gene, which encodes a sterol-biosynthetic enzyme of Saccharomyces cerevisiae. Specifically, the discovery of sterol-mediated feedback control of ERG10 transcription is reported. Deletion analysis of the ERG10 promoter region identified sequences involved in the expression of ERG10. This regulatory axis appeared to involve sterol levels, as a late block in the pathway that depletes sterol, but not nonsterol isoprenoids, was able to elicit the regulatory response.