Chromosomal Assignment of Human Nuclear Envelope Protein Genes LMNA, LMNB1, and LBR by Fluorescencein SituHybridization

We have used fluorescencein situhybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2–q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3–q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.     

High chromosome numbers in Mojavean and Sonoran desert Atriplex canescens (Chenopodiaceae)

Cytological, flavonoid, and morphological data are provided for several varieties of the shrubby species Atriplex canescens (Pursh) Nutt. (x = 9) (fourwing saltbush) in the Mojavean and Sonoran deserts of southwestern United States and northern Mexico. These include var. linearis (S. Wats.) Munz (2x); var. angustifolia (Torr.) S. Wats. (2x, 4x); var. occidentalis (Torr. & Frem.) Welsh & Stutz (4x, 6x), the common variety; var. laciniata Parish (12x); and var. macilenta Jeps. (12x). Atriplex canescens var. grandidentatum Stutz & Sanderson (20x) is newly described. An autoploid origin from 12x var. laciniata is suggested for the 14x and 20x polyploids, through unreduced gametes. Founder populations of odd-ploid products arising during such a sequence of events could probably have returned to even-ploidy through genetic segregation and the rapid elimination of aneuploids. Morphological characters suggest an origin for 12x var. laciniata by interspecific hybridization of var. occidentalis with A. polycarpa (Torr.) S. Wats.     

SEXUAL SELECTION IN AMERICAN TOADS: A TEST OF A GOOD-GENES HYPOTHESIS

Adaptive mate choice in species lacking male resource control and/or paternal care might be maintained by selection because preferred males sire genetically superior offspring. For such a process to occur, some male phenotypic trait(s) must both reliably indicate male genetic quality and influence the pattern of mate choice by females. In American toads, Bufo americanus, male body length has been documented to influence female mating patterns: females usually mate with males that are larger than average. However, the relationship between male size and male genetic quality is unknown. We conducted a controlled breeding experiment using 48 sires and 19 dams to determine if larger males sire offspring with superior larval performance characteristics (greater survival to metamorphosis, larger mass at metamorphosis, and earlier metamorphosis). We also aged each sire to test the hypothesis that older males are, on average, genetically superior to younger males. We crossed each female with three sires representing three body size categories (mean and 1 SD ± mean snout-ischium length). Hatchlings (500 from each cross) were reared to metamorphosis in seminatural ponds in the field. Metamorph weight (log transformed) and age at metamorphosis showed significant heritability and were genetically correlated with each other. Hence, sires differed in genetic quality. However, none of the three measures of offspring performance was correlated with sire body size or age. Thus, we obtained no support for the prediction that sire body size or age is related to genetic quality.     

Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas hydrophila.

Strain C5.84 is a Tn5-751 insertion mutant of Aeromonas hydrophila which is unable to secrete extracellular proteins, instead accumulating them in the periplasm (B. Jiang and S.P. Howard, J. Bacteriol. 173:1241-1249, 1991). A 3.5-kb BglII fragment which complements this mutation was isolated from the chromosome of the parent strain. Analysis of this fragment revealed an operon-like structure with two complete genes, exeA and exeB, a functional promoter 5' to the exeA gene, and a 13-bp inverted repeat immediately 3' to the exeB gene. Although the transposon had inserted in exeA, provision of a wild-type copy of this gene alone in trans did not restore competence for export to C5.84. Complementation required the presence of both exeA and exeB, and marker exchange mutagenesis confirmed the requirement for both gene products for secretion. In vitro expression as well as analysis of the deduced amino acid sequence of ExeA indicated that it is a hydrophilic 60-kDa protein with a consensus ATP binding site. ExeB is a 25-kDa basic protein which shares limited homology with PulB, a protein of unknown function associated with the maltose regulon of Klebsiella oxytoca, and OutB, a protein which has been shown to be required for efficient secretion in Erwinia chrysanthemi. The hydrophilic character of these proteins and preliminary localization studies suggested that they are anchored to the inner membrane. These results demonstrate the involvement of a second operon encoding a putative ATP-binding protein in the secretion of extracellular proteins from gram-negative bacteria and further suggest that the cytoplasmic compartment may play a greater role in protein translocation across the outer membrane from the periplasm than previously thought.     

The force exerted by a single kinesin molecule against a viscous load.

Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising the viscosity of the buffer solution. When the viscosity of the solution used in the low density motility assay was increased approximately 100-fold through addition of polysaccharides and polypeptides, the longer microtubules, which experienced a larger drag force from the fluid, moved more slowly than the shorter ones. The speed of movement of a microtubule depended linearly on the drag force loading the motor. At the lowest kinesin density, where dilution experiments indicated that the movement was caused by a single kinesin molecule, extrapolation of the linear relationship yielded a maximum time-averaged drag force of 4.2 +/- 0.5 pN per motor (mean +/- experimental SE). The magnitude of the force argues against one type of "ratchet" model in which the motor is hypothesized to rectify the diffusion of the microtubule; at high viscosity, diffusion is too slow to account for the observed speeds. On the other hand, our data are consistent with models in which force is a consequence of strain developed in an elastic element within the motor; these models include a different "ratchet" model (of the type proposed by A. F. Huxley in 1957) as well as "power-stroke" models.     

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.