Critical sequences in the core of the P1 plasmid replication origin.

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.     

Abscisic Acid Response of Corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) Roots and Protoplasts to Lanthanum

Lanthanum ions antagonize calcium and are used as a Ca²⁺ channel blocker but their direct effects are unknown. We investigated lanthanum effects on endogenous abscisic acid (ABA) levels in protoplasts and intact primary roots of Zea mays L. Application of 1 mM La³⁺ reduced primary root elongation, caused swelling of root tips, and essentially doubled the ABA content in intact roots but decreased ABA in root protoplasts in a concentration-dependent manner. Osmotic stress increased ABA level in protoplasts more than in intact roots. Temporal ABA changes in response to La³⁺ treatment indicate that La³⁺ affects root growth at least partially via ABA pathway.     

Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.     

Estimating multilevel logistic regression models when the number of clusters is low: a comparison of different statistical software procedures

Multilevel logistic regression models are increasingly being used to analyze clustered data in medical, public health, epidemiological, and educational research. Procedures for estimating the parameters of such models are available in many statistical software packages. There is currently little evidence on the minimum number of clusters necessary to reliably fit multilevel regression models. We conducted a Monte Carlo study to compare the performance of different statistical software procedures for estimating multilevel logistic regression models when the number of clusters was low. We examined procedures available in BUGS, HLM, R, SAS, and Stata. We found that there were qualitative differences in the performance of different software procedures for estimating multilevel logistic models when the number of clusters was low. Among the likelihood-based procedures, estimation methods based on adaptive Gauss-Hermite approximations to the likelihood (glmer in R and xtlogit in Stata) or adaptive Gaussian quadrature (Proc NLMIXED in SAS) tended to have superior performance for estimating variance components when the number of clusters was small, compared to software procedures based on penalized quasi-likelihood. However, only Bayesian estimation with BUGS allowed for accurate estimation of variance components when there were fewer than 10 clusters. For all statistical software procedures, estimation of variance components tended to be poor when there were only five subjects per cluster, regardless of the number of clusters.