Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements

Horizontal transmission has been well documented as a major mechanism for the dissemination of mariner-like elements (MLEs) among species. Less well understood are mechanisms that limit vertical transmission of MLEs resulting in the "spotty" or discontinuous distribution observed in closely related species. In this article we present evidence that the genome of the common ancestor of the melanogaster species subgroup of Drosophila contained an MLE related to the mellifera (honey bee) subfamily. Horizontal transmission, approximately 3-10 MYA, is strongly suggested by the observation that the sequence of the MLE in Drosophila erecta is 97% identical in nucleotide sequence with that of an MLE in the cat flea, Ctenocephalides felis. The D. erecta MLE has a spotty distribution among species in the melanogaster subgroup. The element has a high copy number in D. erecta and D. orena, a moderate copy number in D. teissieri and D. yakuba, and was apparently lost ("stochastic loss") in the lineage leading to D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In D. erecta, most copies are concentrated in the heterochromatin. Two copies from D. erecta, denoted De12 and De19, were cloned and sequenced, and they appear to be nonfunctional ("vertical inactivation"). It therefore appears that the predominant mode of MLE evolution is vertical inactivation and stochastic loss balanced against occasional reinvasion of lineages by horizontal transmission.      

Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Background  

Nitric oxide and prostaglandin E₂ (PGE₂play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

Introns and reading frames: correlation between splicing sites and their codon positions

Computer analyses of the entire GenBank database were conducted to examine correlation between splicing sites and codon positions in reading frames. Intron insertion patterns (i.e., splicing site locations with respect to codon positions) have been analyzed for all of the 74 codons of all the eukaryote taxonomic groups: primates, rodents mammals, vertebrates, invertebrates, and plants. We found that reading frames are interrupted by an intron at a codon boundary (as opposed to the middle of a codon) significantly more often than expected. This observation is consistent with the exon shuffling hypothesis, because exons that end at codon boundaries can be concatenated without causing a frame shift and thus are evolutionarily advantageous. On the other hand, when introns interrupt at the middles of codons, they exist in between the first and second bases much more frequently than between the second and third bases, despite the fact that boundaries between the first and second bases of codons are generally far more important than those between the second and third bases. The reason for this is not clear and yet to be explained. We also show that the length of an exon is a multiple of 3 more frequently than expected. Furthermore, the total length of two consecutive exons is also more frequently a multiple of 3. All the observations above are consistent with results recently published by Long, Rosenberg, and Gilbert (1995).      

Towards a phylogeny and evolution of Acochlidia (Mollusca: Gastropoda: Opisthobranchia)

The Acochlidia are unique among opisthobranch gastropods in combining extremely high morphological and ecological diversity with modest species diversity. The phylogeny of acochlidians has never been addressed by cladistic means, as their evolution has remained unknown. This study gives a first overview on more than 150 biological and morphological characters that are potentially useful for phylogenetic analysis. Based on 107 characters, a parsimony analysis (PAUP) was performed for all 27 valid acochlidian species together with 11 (plus two) outgroup taxa. The resulting strict consensus tree shows a moderate overall resolution, with at least some bootstrap support for most resolved nodes. The Acochlidia are clearly monophyletic, and originate from an unresolved basal opisthobranch level. The Acochlidia split into the Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia (‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina (Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))). The formerly enigmatic Ganitidae, resembling sacoglossan opisthobranchs by having dagger‐like rachidian radular teeth, are likely to be highly derived microhedylids. The paraphyly of some of the traditionally recognized family level taxa induced a preliminary reclassification. From the phylogenetic hypothesis obtained, we conclude that the acochlidian ancestor was marine mesopsammic. The colonization of limnic systems occurred twice, independently: first in the Caribbean (with the development of the small interstitial Tantulum elegans), and second in the Indo‐Pacific, with a radiation of large‐sized benthic acochlidian species. The evolution of extraordinary reproductive features, such as hypodermic impregnation by a complex copulative aparatus in hedylopsaceans, cutaneous insemination via spermatophores in microhedylaceans, and gonochorism in Microhedylidae s.l. (including Ganitidae), is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 124–154.