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21.
Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration 总被引:14,自引:4,他引:10
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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. 相似文献
22.
Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli 总被引:2,自引:0,他引:2
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.
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23.
24.
Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli 总被引:7,自引:1,他引:6
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.
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25.
Abulencia CB Wyborski DL Garcia JA Podar M Chen W Chang SH Chang HW Watson D Brodie EL Hazen TC Keller M 《Applied and environmental microbiology》2006,72(5):3291-3301
Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173. 相似文献
26.
TT Chowdhury S Arghandawi J Brand OO Akanji DL Bader DM Salter DA Lee 《Arthritis research & therapy》2008,10(2):R35
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. 相似文献27.
Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation.
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Eukaryotic expression vectors designed to produce E. coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells. 相似文献
28.
Introns and reading frames: correlation between splicing sites and their codon positions 总被引:4,自引:1,他引:3
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).
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29.
Clifford DL Folmes D Kent Arrell Jelena Zlatkovic-Lindor Almudena Martinez-Fernandez Carmen Perez-Terzic Timothy J Nelson Andre Terzic 《Cell cycle (Georgetown, Tex.)》2013,12(15):2355-2365
Nuclear reprogramming resets differentiated tissue to generate induced pluripotent stem (iPS) cells. While genomic attributes underlying reacquisition of the embryonic-like state have been delineated, less is known regarding the metabolic dynamics underscoring induction of pluripotency. Metabolomic profiling of fibroblasts vs. iPS cells demonstrated nuclear reprogramming-associated induction of glycolysis, realized through augmented utilization of glucose and accumulation of lactate. Real-time assessment unmasked downregulated mitochondrial reserve capacity and ATP turnover correlating with pluripotent induction. Reduction in oxygen consumption and acceleration of extracellular acidification rates represent high-throughput markers of the transition from oxidative to glycolytic metabolism, characterizing stemness acquisition. The bioenergetic transition was supported by proteome remodeling, whereby 441 proteins were altered between fibroblasts and derived iPS cells. Systems analysis revealed overrepresented canonical pathways and interactome-associated biological processes predicting differential metabolic behavior in response to reprogramming stimuli, including upregulation of glycolysis, purine, arginine, proline, ribonucleoside and ribonucleotide metabolism, and biopolymer and macromolecular catabolism, with concomitant downregulation of oxidative phosphorylation, phosphate metabolism regulation, and precursor biosynthesis processes, prioritizing the impact of energy metabolism within the hierarchy of nuclear reprogramming. Thus, metabolome and metaboproteome remodeling is integral for induction of pluripotency, expanding on the genetic and epigenetic requirements for cell fate manipulation. 相似文献
30.