Bacterial RNA isolation with one hour centrifugation in a table-top ultracentrifuge.

A procedure for the rapid preparation of cesium-chloride purified RNA from E. coli and the cyanobacterium Synechococcus sp. PCC7942 is described. Cells are lysed in modified sucrose, Triton X-100, EDTA, Tris buffer with phenol/chloroform. The cleared lysate is extracted further with phenol/chloroform and RNA is peleted by centrifugation through a 5.7 M CsCl cushion. High quality RNA can be prepared in three hours using this procedure.     

Sequence comparison with concave weighting functions

We consider efficient methods for computing a difference metric between two sequences of symbols, where the cost of an operation to insert or delete a block of symbols is a concave function of the block's length. Alternatively, sequences can be optimally aligned when gap penalties are a concave function of the gap length. Two algorithms based on the ‘candidate list paradigm’ first used by Waterman (1984) are presented. The first computes significantly more parsimonious candidate lists than Waterman's method. The second method refines the first to the point of guaranteeingO(N ² lgN) worst-case time complexity, and under certain conditionsO(N ²). Experimental data show how various properties of the comparison problem affect the methods' relative performance. A number of extensions are discussed, among them a technique for constructing optimal alignments inO(N) space in expectation. This variation gives a practical method for comparing long amino sequences on a small computer. This work was supported in part by NSF Grant DCR-8511455.     

Brain blotting: a method to detect multiple DNA copies in specific brain regions

We developed a method to detect multiple DNA copies (both cellular and viral) in specific brain regions by blotting thick frozen sections onto nylon membranes. This was achieved by "printing" the frozen sections on standard blotting paper immediately after cryotome sectioning and performing blotting according to the standard Southern technique. A "replica" of the blotted section was obtained by keeping on the glass slide the next frozen section cut, which was then stained for conventional histopathological analysis and the cell nuclei counted to give an estimate of the total amount of DNA present in each section. The blotted membranes were then denatured and hybridized with a nick-translated Alu probe either at 42 degrees C with 50% formamide or at 68 degrees C without formamide. Brain sections from mice infected with Herpes simplex virus 1 (HSV1), blotted and hybridized with a nick-translated HSV1 probe, clearly showed the focal nature of the Herpes simplex infection, which was also demonstrated immunohistologically using a virus specific antiserum. This method of DNA detection, conveniently modified, might also be used to detect nuclear and cytoplasmic RNAs in specific coronal sections of whole brain before localization at high power by standard in situ techniques.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.     

Optimal alignments in linear space

Space, not time, is often the limiting factor when computingoptimal sequence alignments, and a number of recent papers inthe biology literature have proposed space-saving strategies.However, a 1975 computer science paper by Hirschberg presenteda method that is superior to the new proposals, both in theoryand in practice. The goal of this paper is to give Hirschberg'sidea the visibility it deserves by developing a linear-spaceversion of Gotoh's algorithm, which accommodates affine gappenalties. A portable C-software package implementing this algorithmis available on the BIONET free of charge. Received on October 14, 1987; accepted on December 19, 1987     

Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b

Several years ago, prochlorophyte picoplankton were discovered in the N. Atlantic. They have since been found to be abundant within the euphotic zone of the world's tropical and temperate oceans. The cells are extremely small, lack phycobiliproteins, and contain divinyl chlorophyll a and b as their primary photosynthetic pigments. Phylogenies constructed from DNA sequence data indicate that these cells are more closely related to a cluster of marine cyanobacteria than to their prochlorophyte relatives Prochlorothrix and Prochloron. Several strains of this organism have recently been brought into culture, and herewith are given the name Prochlorococcus marinus.     

Maternal origin of deletion 15q11–13 in 25/25 cases of Angelman syndrome

Summary About half of the cases of Angelman syndrome arise from deletions of chromosome band 15q12. In 25 cases we have been able to determine the parental origin of the deletion and, in line with other reported cases, we have found the deletion to be of maternal origin. There were no exceptions. The parental origin was determined using cytogenetic markers in 13 of the cases, in nine by using the pattern of inheritance of restriction fragment length polymorphisms, and in three using both techniques.     

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.     

The limits of the cellular capacity to mediate an estrogen response.

While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.