Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 × 10−5 to 4 × 10−5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5–28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation. Typical cases have 3/4 finger and 4/5 toe syndactyly, with a duplicated digit in the syndactylous web, but incomplete penetrance and variable expressivity are common. The condition has recently been shown to be caused by expansions of an imperfect trinucleotide repeat sequence encoding a 15-residue polyalanine tract in HOXD13. We have studied 16 new and 4 previously published SPD families, with between 7 and 14 extra residues in the tract, to analyze the molecular basis for the observed variation in phenotype. Although there is no evidence of change in expansion size within families, even over six generations, there is a highly significant increase in the penetrance and severity of phenotype with increasing expansion size, affecting both hands (P = 0.012) and feet (P < 0.00005). Affected individuals from a family with a 14-alanine expansion, the largest so far reported, all have a strikingly similar and unusually severe limb phenotype, involving the first digits and distal carpals. Affected males from this family also have hypospadias, not previously described in SPD, but consistent with HOXD13 expression in the developing genital tubercle. The remarkable correlation between phenotype and expansion size suggests that expansion of the tract leads to a specific gain of function in the mutant HOXD13 protein, and has interesting implications for the role of polyalanine tracts in the control of transcription.

The asymptotic distribution of canonical correlations and vectors in higher-order cointegrated models

The study of the large-sample distribution of the canonical correlations and variates in cointegrated models is extended from the first-order autoregression model to autoregression of any (finite) order. The cointegrated process considered here is nonstationary in some dimensions and stationary in some other directions, but the first difference (the “error-correction form”) is stationary. The asymptotic distribution of the canonical correlations between the first differences and the predictor variables as well as the corresponding canonical variables is obtained under the assumption that the process is Gaussian. The method of analysis is similar to that used for the first-order process.

Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.