Molecular detection of chromosomal translocations that disrupt the putative retinoblastoma susceptibility locus.

A candidate DNA sequence with many of the properties predicted for the retinoblastoma susceptibility (RB1) locus has been cloned (S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature [London] 323:643-645, 1986). The large size of this gene (ca. 200 kilobases [kb]) and its multiple dispersed exons (Wiggs et al., N. Engl. J. Med. 318:151-157, 1988) complicate molecular screening strategies important in prenatal and presymptomatic diagnosis and in carrier detection. Here we used field inversion gel electrophoresis (FIGE) to construct a restriction map of approximately 1,000 kb of DNA surrounding the RB1 locus and to detect the translocation breakpoints in three retinoblastoma patients. DNA probes from either the 5' or 3' end of the gene were used to detect a 250-kb EagI restriction fragment in DNA from unaffected individuals. Both probes identified an additional hybridizing fragment in the DNA from each patient, permitting the breakpoints in all three to be mapped within the cloned RB1 gene. Analysis of the breakpoint in one translocation cell line allowed the RB1 gene to be oriented with its 5' end toward the centromere. The 5' end of the gene also appeared to be associated with a clustering of sites for several infrequently cleaving restriction enzymes, indicating the presence of an HpaII tiny fragment island. The detection and mapping of the translocation breakpoints of all three retinoblastoma patients to within the putative RB1 gene substantiated the authenticity of this candidate sequence and demonstrated the utility of FIGE in detecting chromosomal rearrangements affecting this locus.     

A reversible arrest point in the late G1 phase of the mammalian cell cycle

The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.     

Olfactory memory: the long and short of it

It has been proposed that memory for odors does not have a short-term (or working) memory system. The distinction between short- and long- term memory in other sensory modalities has been generally supported by three main lines of evidence: capacity differences between the proposed systems, evidence of differential coding, and differential memory losses in neuropsychological patients. The present paper examines these issues in an effort to establish a similar distinction for the memory of olfactory stimuli. Each of these lines of evidence is examined in relation to the literature on olfactory memory. Based on this examination, it seems that there is at least preliminary support from each of these lines of evidence to advocate a distinction between a long- and short-term memory for olfactory stimuli. Emphasis is placed upon the qualitative similarity of olfactory memory to other memory systems. This similarity is further highlighted through an examination of the literature pertinent to serial position effects in memory for olfactory stimuli.      

A New Framework to Accurately Quantify Soil Bacterial Community Diversity from DGGE

Denaturing gradient gel electrophoresis (DGGE) has been and remains extensively used to assess and monitor the effects of various treatments on soil bacterial communities. Considering only abundant phylotypes, the diversity estimates produced by this technique have been proven to be uncorrelated to true community diversity. The aim of this paper was to develop a framework to estimate a community’s true diversity from DGGE. Developed using in silico DGGE profiles generated from published pyrosequencing datasets, this framework elongates the rank-abundance distributions (RADs) drawn by band quantification using the peak-to-signal ratio (PSR) parameter, which was proven to be related to bacterial richness. The ability to compare DGGE-based diversity estimates to the true diversity of communities led to a unique opportunity to identify potential pitfalls when analyzing DGGE gels with commercial analysis software programs and gain insight into the process of DNA band clustering in the profiles. Bacterial diversity was compared through richness, Shannon, and Simpson’s 1/D indices. Intermediate results demonstrated that, even though commercial gel analysis software programs were unable to produce consistent results throughout all samples, a newly developed Matlab-based framework unraveled the dominance profiles of communities from band quantification. Elongating these partial RADs using the PSRs extracted from the DGGE profiles chiefly made it possible to accurately estimate the true diversity of communities. For all the samples analyzed, the estimated Shannon and Simpson’s 1/D were accurate at ±10 %. Richness estimations were less accurate, ranging from ?11 to 31 % of the expected values. The framework showed great potential to study the structure and diversity of soil bacterial communities.     

A new class of reversible cell cycle inhibitors

The effects of three compounds on the cell cycle of HL-60 promyeloid leukemia cells has been examined. Ciclopirox olamine, an antifungal agent, and the compound Hoechst 768159 reversibly block the cell cycle at a point occurring roughly 1 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which acts in early S phase. Similar results are also obtained with the compound mimosine, a plant amino acid. Based on these data, it is concluded that all three agents inhibit cell cycle traverse at or very near the G1/S phase boundary and identify a previously undefined reversible cell cycle arrest point.