Highly conserved regimes of neighbor-base-dependent mutation generated the background primary-structural heterogeneities along vertebrate chromosomes.

The content of guanine+cytosine varies markedly along the chromosomes of homeotherms and great effort has been devoted to studying this heterogeneity and its biological implications. Already before the DNA-sequencing era, however, it was established that the dinucleotides in the DNA of mammals in particular, and of most organisms in general, show striking over- and under-representations that cannot be explained by the base composition. Here we show that in the coding regions of vertebrates both GC content and codon occurrences are strongly correlated with such "motif preferences" even though we quantify the latter using an index that is not affected by the base composition, codon usage, and protein-sequence encoding. These correlations are likely to be the result of the long-term shaping of the primary structure of genic and non-genic DNA by a regime of mutation of which central features have been maintained by natural selection. We find indeed that these preferences are conserved in vertebrates even more rigidly than codon occurrences and we show that the occurrence-preference correlations are stronger in intronic and non-genic DNA, with the R(2)s reaching 99% when GC content is approximately 0.5. The mutation regime appears to be characterized by rates that depend markedly on the bases present at the site preceding and at that following each mutating site, because when we estimate such rates of neighbor-base-dependent mutation (NBDM) from substitutions retrieved from alignments of coding, intronic, and non-genic mammalian DNA sorted and grouped by GC content, they suffice to simulate DNA sequences in which motif occurrences and preferences as well as the correlations of motif preferences with GC content and with motif occurrences, are very similar to the mammalian ones. The best fit, however, is obtained with NBDM regimes lacking strand effects, which indicates that over the long term NBDM switches strands in the germline as one would expect for effects due to loosely contained background transcription. Finally, we show that human coding regions are less mutable under the estimated NBDM regimes than under matched context-independent mutation and that this entails marked differences between the spectra of amino-acid mutations that either mutation regime should generate. In the Discussion we examine the mechanisms likely to underlie NBDM heterogeneity along chromosomes and propose that it reflects how the diversity and activity of lesion-bypass polymerases (LBPs) track the landscapes of scheduled and non-scheduled genome repair, replication, and transcription during the cell cycle. We conclude that the primary structure of vertebrate genic DNA at and below the trinucleotide level has been governed over the long term by highly conserved regimes of NBDM which should be under direct natural selection because they alter drastically missense-mutation rates and hence the somatic and the germline mutational loads. Therefore, the non-coding DNA of vertebrates may have been shaped by NBDM only epiphenomenally, with non-genic DNA being affected mainly when found in the proximity of genes.

Topical application of laminin-332 to diabetic mouse wounds.

BACKGROUND: Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration. OBJECTIVE: To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds. METHODS: LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured. RESULTS: Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05). CONCLUSION: The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing.

Mammalian mutation pressure, synonymous codon choice, and mRNA degradation.

The usage of synonymous codons (SCs) in mammalian genes is highly correlated with local base composition and is therefore thought to be determined by mutation pressure. The usage is nonetheless structured. For instance, mammals share with Saccharomyces and Drosophila most preferences for the C-ending over the G-ending codon (or vice versa) within each fourfold-degenerate SC family and the fact that their SCs are placed along coding regions in ways that minimize the number of T|A and C|G dinucleotides ("|" being the codon boundary). TA and CG underrepresentations are observed everywhere in the mammalian genome affecting the SC usage, the amino acid composition of proteins, and the primary structure of introns and noncoding DNA. While the rarity of CG is ascribed to the high mutability of this dinucleotide, the rarity of TA in coding regions is considered adaptive because UA dinucleotides are cleaved by endoribonucleases. Here we present in vivo experimental evidence indicating that the number of T|A and/or C|G dinucleotides of a human gene can affect strongly the expression level and degradation of its mRNA. Our results are consistent with indirect evidence produced by other workers and with the detailed work that has been devoted to characterize UA cleavage in vitro and in vivo. We conclude that SC choice can influence strongly mRNA function and gene expression through effects not directly related to the codon-anticodon interaction. These effects should constrain heavily the nucleotide motif composition of the most abundant mRNAs in the transcriptome, in particular, their SC usage, a usage that must be reflected by cellular tRNA concentrations and thus defines for all other genes which SCs are translated fastest and most accurately. Furthermore, the need to avoid such effects genome-wide appears serious enough to have favored the evolution of biases in context-dependent mutation that reduce the occurrence of intrinsically unfavorable motifs, and/or, when possible, to have induced the molecular machinery mediating such effects to rely opportunistically on already existing motif rarities and abundances. This may explain why nucleotide motif preferences are very similar in transcribed and nontranscribed mammalian DNA even though the preferences appear to be adaptive only in transcribed DNA.