Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy.

We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253. These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA. Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy. Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion. Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo. C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene. The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12. These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Action of Brefeldin A on Amphibian Neurons: Passage of Newly Synthesized Proteins Through the Golgi Complex Is Not Required for Continued Fast Organelle Transport in Axons

Abstract: The relation between the availability of newly synthesized protein and lipid and the axonal transport of optically detectable organelles was examined in peripheral nerve preparations of amphibia (Rana catesbeiana and Xenopus laevis) in which intracellular traffic from the endo-plasmic reticulum to the Golgi complex was inhibited with brefeldin A (BFA). Accumulation of fast-transported radio-labeled protein or phospholipid proximal to a sciatic nerve ligature was monitored in vitro in preparations of dorsal root ganglia and sciatic nerve. Organelle transport was examined by computer-enhanced video microscopy of single myelinated axons. BFA reduced the amount of radiolabeled protein and lipid entering the fast-transport system of the axon without affecting either the synthesis or the transport rate of these molecules. The time course of the effect of BFA on axonal transport is consistent with an action at an early step in the intrasomal pathway, and with its action being related to the observed rapid (<1 h) disassembly of the Golgi complex. At a concentration of BFA that reduced fast-transported protein by >95%, no effect was observed on the flux or velocity of anterograde or retrograde organelle transport in axons for at least 20 h. Bidirectional axonal transport of organelles was similarly unaffected following suppression of protein synthesis by >99%. The findings suggest that the anterograde flux of transport organelles is not critically dependent on a supply of newly synthesized membrane precursors. The possibilities are considered that anterograde organelles normally arise from membrane components supplied from a post-Golgi storage pool, as well as from recycled retrograde organelles.     

Longitudinal patterns of dissolved organic carbon concentration and suspended bacterial density along a blackwater river

Dissolved organic carbon (DOC) is the dominant form of carbon in transport in blackwater rivers, and bacteria are the major biological agents of its utilization. This study describes longitudinal patterns in DOC concentration and relates them to suspended bacterial populations in the channel. Concentrations of total DOC, three molecular weight fractions, and bacterial numbers were determined at 12 sites along the Ogeechee River in 1985–1986 and 1989 during periods of low and high discharge. Suspended bacterial populations were compared with DOC concentrations to determine if differences in bacterial abundance were related to longitudinal patterns of DOC concentration. Three distinct longitudinal patterns were observed: (1) The longitudinal pattern followed by both total and intermediate molecular weight DOC concentrations was a linear function of the geographic distance along the river. (2) During low flow conditions, there was a high degree of correspondence between patterns of bacterial numbers and low MW DOC (< 1000 apparent MW). (3) During periods of high discharge, the proportion of high (> 10,000) and intermediate (1000–10,000) MW fractions increased, and there was no longer a clear relationship between bacterial cells and low MW DOC.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

BRCA1 maps proximal to D17S579 on chromosome 17q21 by genetic analysis

Previous studies have demonstrated linkage between early-onset breast cancer and ovarian cancer and genetic markers on chromosome 17q21. These markers define the location of a gene (BRCA1) which appears to be inherited as an autosomal dominant susceptibility allele. We analyzed five families with multiple affected individuals for evidence of linkage to the BRCA1 region. Two of the five families appear to be linked to BRCA1. One apparently linked family contains critical recombinants, suggesting that the gene is proximal to the marker D17S579 (Mfd188). These findings are consistent with the maximum-likelihood position estimated by the Breast Cancer Linkage Consortium and with recombination events detected in other linked families. Linkage analysis was greatly aided by PCR-based analysis of paraffin-embedded normal breast tissue from deceased family members, demonstrating the feasibility and importance of this approach. One of the two families with evidence of linkage between breast cancer and genetic markers flanking BRCA1 represents the first such family of African-American descent to be reported in detail.     

Topography of short portal vessels in the rat pituitary gland: a scanning electron-microscopic and morphometric study of corrosion cast replicas

We applied scanning electron microscopy combined with imaging and morphometric techniques to analyze the dorsal topography and morphology of short portal vessels linking the capillary beds of the pituitary neural and anterior lobes in adult male albino rats. The pituitary microvasculature was replicated by intracarotid injection of Batson's No. 17 compound producing plastic casts that were advantageous for comprehensive morphometric analyses using an imaging device. The analysis revealed the existence of two types of portal vessels having quantitatively different morphological properties. The bilateral venular plexus of 3–4 vessels located at the base of the infundibular stalk (each venule measuring 300 m in length and 32 m in diameter) appears to be the major part of the short portal system in the dorsum of the rat pituitary gland. Narrower capillary-like shunt vessels (6.8 m in diameter), of about the same length as the venules, were situated throughout other subregions of the intermediate lobe cleft. The short portal vessels of both types made direct anastomoses with the capillary networks in the neural and anterior lobes. The neural lobe capillaries were twice as numerous (1324 per mm²), and only half as wide (6.2 m), as the sinusoidal capillaries in the anterior lobe (density of 637 per mm²; diameter of 13.7 m). The topographical position of the portal venular system suggests that the caudolateral subregions of the pituitary neural and anterior lobes have a functional relationship dependent on rapid interlobe transfer of neurohumoral factors such as hormones via the portal blood. This process appears to be supplemented throughout the rest of the cleft between the two lobes by a small number of capillary shunts that supply the epithelial cell lobules of the intermediate lobe in situ. The findings collectively indicate that this portal system provides a constant stream of neurohumoral information that is shared moment-by-moment between the pituitary neural and anterior lobes.     

Latent group detection in functional partially linear regression models

In this paper, we propose a functional partially linear regression model with latent group structures to accommodate the heterogeneous relationship between a scalar response and functional covariates. The proposed model is motivated by a salinity tolerance study of barley families, whose main objective is to detect salinity tolerant barley plants. Our model is flexible, allowing for heterogeneous functional coefficients while being efficient by pooling information within a group for estimation. We develop an algorithm in the spirit of the K-means clustering to identify latent groups of the subjects under study. We establish the consistency of the proposed estimator, derive the convergence rate and the asymptotic distribution, and develop inference procedures. We show by simulation studies that the proposed method has higher accuracy for recovering latent groups and for estimating the functional coefficients than existing methods. The analysis of the barley data shows that the proposed method can help identify groups of barley families with different salinity tolerant abilities.