YAC cloning of DNA embedded in an agarose matrix

Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.     

A model for 8-10 Hz spindling in interconnected thalamic relay and reticularis neurons.

We investigated a simplified model of a thalamocortical cell and a reticular thalamic cell interconnected with excitatory and inhibitory synapses, based on Hodgkin-Huxley type kinetics. The intrinsic oscillatory properties of the model cells were similar to those observed from single cells in vitro. When synaptic interactions were included, spindle oscillations were observed consisting of sequences of rhythmic oscillations at 8-10 Hz separated by silent periods of 8-40 s. The model suggests that Ca2+ regulation of lh channels may be responsible for the waxing and waning of spindles and that the reticular cell shapes the 10-Hz rhythmicity. The model also predicts that the kinetics of gamma-aminobutyric acid inhibitory postsynaptic potentials as well as the intrinsic properties of reticular cells are critical in determining the frequency of spindle rhythmicity.     

A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes themale sterile 14 gene and a hotspot for recombination

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized onems locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that thems14 locus is located in a ～300 kb region. We have identified a YAC clone with an insert size of ～610 kb that contains thems14-linked markers, reflects the organization of the physical map and therefore most probably contains thems14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from ～105–140 kb/cM to less than 24 kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.     

Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor

In the filamentous bacterium Streptomyces coelicolor , the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 μm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 μm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants ( whiG , whiH and whiB ), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.     

Auditory sensitivity and psychophysical tuning curves in the elephant nose fish,Gnathonemus petersii

Summary Hearing sensitivity and psychophysical tuning curves were determined for the mormyridGnathonemus petersii. Pure tone hearing thresholds were determined from 100 Hz to 2,500 Hz, with best sensitivity being about –31 dB (re: 1 dyne/ cm²) from 300 Hz to 1,000 Hz. In order to determine frequency tuning of the auditory system, psychophysical tuning curves (PTC's) were measured with the masker presented simultaneously with, or just ahead of, the 500 Hz test signal. The sound level for different frequencies needed to just mask the test tone were determined from 100 to 800 Hz. Maximum masking occurred in both forward and simultaneous conditions when the masker and the test tone were at the same frequency. As the masker was moved in frequency from 500 Hz, higher sound levels of maskers were needed to afford masking of the test tone. The data were similar in simultaneous and forward masking, with theQ _{10 dB}, a measure of sharpness of tuning, being about 5 in both cases. Data were compared for other species for which behavioral thresholds and PTC's are available.Gnathonemus hears about as wide a range of frequencies as the goldfish,Carassius auratus, although the PTC's for the two species are strikingly different. The PTC's forGnathonemus resemble those determined in a forward-masking paradigm for the clown knife fish,Notopterus chitala, even thoughGnathonemus has a wider hearing bandwidth.Abbreviations AM amplitude modulated - EOD electric organ discharge - PTC psychophysical tuning curve     

Effects of chronic hyperinsulinaemia on hepatic enzymes involved in lipogenesis and carbohydrate metabolism in the young rat.

Chronic (6 days) hyperinsulinaemia in young rats produced lower blood glucose concentrations and augmented body- and liver-weight gain. The insulin-treated rats had increased hepatic activities of citrate-cleavage enzyme, 'malic' enzyme and high-substrate (6.6 mM-phosphoenolpyruvate) pyruvate kinase, and decreased glucose 6-phosphatase. There were no changes in activities of phosphoenolpyruvate carboxykinase, phosphofructokinase, low-substrate (1.3 mM-phosphoenolpyruvate) pyruvate kinase, glucokinase and hexokinase.