Prediction of rho-independent transcriptional terminators in Escherichia coli.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3'-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted 'orphan' promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

The subcellular localization of lipid in myelin-free axonal preparations.

Bovine myelin-free axonal preparations were subjected to a series of washes designed to partition membranes from other cellular components initially present in these preparations. These washes were composed entirely of membranous structures, essentially free of neurofilament protein subunits, and contained high specific activity of acetylcholinesterase, an axolemma-specific enzyme. The distribution of acetylcholinesterase in the washes paralleled the distribution of lipid and the lipid composition of these washes closely resembled that of bovine axolemma-enriched fractions. In addition, acetylcholinesterase, lipid and galactocerebroside were histo- and immunohistochemically localized on similar structures in the starting material. Our results demonstrate that some of the lipid in myelin-free axonal preparations may be accounted for by axolemma.

Characterization of a V kappa family in Mus musculus castaneus: expansion at the subset level.

We have examined the same kappa chain variable (V kappa) region family in several mouse species in order to observe short-term, incremental change at immunoglobulin (Ig) multigene loci. In the present study, the Igk-V24 family has been characterized in a Mus m. castaneus colony derived from individuals originating in Thailand and compared to the same family in Mus m. domesticus (BALB/c) and Mus pahari, representing about 1-2 and about 5-9 million years of evolution, respectively. Southern hybridization of genomic DNA with a probe encoding the prototype Igk-V24 coding region reveals restriction fragment patterns indicative of two distinct M. m. castaneus haplotypes. These haplotypes appear to result from an unequal recombination between similar gene arrays, as their restriction patterns are unique but contain many common fragments. The complexity of these patterns indicates a marked expansion in the Igk-V24 family of M. m. castaneus relative to BALB/c and M. pahari. Additional analyses using probes specific for individual subsets demonstrate that the expansion is not general throughout the entire family, but is restricted to particular subsets and therefore to relatively short chromosomal segments. One subset alone accounts for most of the expansion and comprises over 40% of the entire M. m. castaneus family. The wide range of Igk-V24 family complexity seen among M. m. castaneus, M. m. domesticus, and M. pahari, as well as among the different M. m. castaneus family subsets, suggests a model of random evolution in V kappa family copy number rather than one which is selective.

Characterization of a V kappa family in Mus musculus castaneus: sequence analysis.

To examine genetic variation at immunoglobulin (Ig) multigene loci over short spans of evolutionary time, we have compared members of an Ig kappa chain variable (V kappa) region family from several mouse species. In this study, seven unique Igk-V24 family members have been isolated from Mus m. castaneus and characterized by nucleotide sequence determination for comparison to their counterparts in Mus m. domesticus (BALB/c), and Mus pahari, representing 1-2 million years of evolution in the former case and 5-8 million years in the latter. Parsimony, together with evolutionary distances calculated for various pairs of Igk-V24 family coding regions, relate all family members to a common progenitor existing roughly 24 million years ago (Mya). A significant portion of the M. m. castaneus family consists of pseudogene segments in various degrees of progressive degeneration. The substitution patterns and divergence rates for all gene segments are characteristic of their respective subsets, especially in the areas flanking the coding regions. Complex and variable patterns of diversity are seen in potentially expressed coding regions, which appear to reflect quite different selective pressures on various subregions within the V kappa protein domain. These results indicate that evolutionary pressures are operating at the level of family subsets, their individual members, and subregions within similar molecules.