Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.     

Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.     

The Complete Mitochondrial Genome Sequence of the Hornwort <Emphasis Type="Italic">Megaceros</Emphasis><Emphasis Type="Italic">aenigmaticus</Emphasis> Shows a Mixed Mode of Conservative Yet Dynamic Evolution in Early Land Plant Mitochondrial Genomes

Land plants possess some of the most unusual mitochondrial genomes among eukaryotes. However, in early land plants these genomes resemble those of green and red algae or early eukaryotes. The question of when during land plant evolution the dramatic change in mtDNAs occurred remains unanswered. Here we report the first completely sequenced mitochondrial genome of the hornwort, Megaceros aenigmaticus, a member of the sister group of vascular plants. It is a circular molecule of 184,908 base pairs, with 32 protein genes, 3 rRNA genes, 17 tRNA genes, and 30 group II introns. The genome contains many genes arranged in the same order as in those of a liverwort, a moss, several green and red algae, and Reclinomonas americana, an early-branching eukaryote with the most ancestral form of mtDNA. In particular, the gene order between mtDNAs of the hornwort and Physcomitrella patens (moss) differs by only 8 inversions and translocations. However, the hornwort mtDNA possesses 4 derived features relative to green alga mtDNAs—increased genome size, RNA editing, intron gains, and gene losses—which were all likely acquired during the origin and early evolution of land plants. Overall, this genome and those of other 2 bryophytes show that mitochondrial genomes in early land plants, unlike their seed plant counterparts, exhibit a mixed mode of conservative yet dynamic evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Libo Li and Bin Wang contributed equally to this work.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

Conservation, Rearrangement, and Deletion of Gene Pairs During the Evolution of Four Grass Genomes

Gene order and content differ among homologous regions of closely related genomes. Similarities in the expression profiles of physically adjacent genes suggest that the proper functioning of these genes depends on maintaining a specific position relative to each other. To better understand the results of the interaction of these two genomic forces, convergent, divergent, and tandem gene pairs in rice and sorghum, as well as their homologs in rice, sorghum, maize, and Brachypodium were analyzed. The status of each pair in all four species: whether it was conserved, inverted, rearranged, or missing homologs was determined. We observed that divergent gene pairs had lower rates of conservation than convergent or tandem pairs, but higher rates of rearranged pairs and missing homologs in maize than in any other species. We also discovered species-specific gene pairs in rice and sorghum. In rice, gene pairs with strongly correlated expression levels were conserved significantly more often than those with little or no correlation. We assigned three types of gene pair to one of 14 possible evolutionary history categories to uncover their evolutionary dynamics during the evolution of grass genomes.     

The Relationship Between the Rate of Molecular Evolution and the Rate of Genome Rearrangement in Animal Mitochondrial Genomes

Evolution of mitochondrial genes is far from clock-like. The substitution rate varies considerably between species, and there are many species that have a significantly increased rate with respect to their close relatives. There is also considerable variation among species in the rate of gene order rearrangement. Using a set of 55 complete arthropod mitochondrial genomes, we estimate the evolutionary distance from the common ancestor to each species using protein sequences, tRNA sequences, and breakpoint distances (a measure of the degree of genome rearrangement). All these distance measures are correlated. We use relative rate tests to compare pairs of related species in several animal phyla. In the majority of cases, the species with the more highly rearranged genome also has a significantly higher rate of sequence evolution. Species with higher amino acid substitution rates in mitochondria also have more variable amino acid composition in response to mutation pressure. We discuss the possible causes of variation in rates of sequence evolution and gene rearrangement among species and the possible reasons for the observed correlation between the two rates. [Reviewing Editor: Dr. David Pollock]     

Gene rearrangements in gekkonid mitochondrial genomes with shuffling,loss, and reassignment of tRNA genes

Background

Vertebrate mitochondrial genomes (mitogenomes) are 16–18 kbp double-stranded circular DNAs that encode a set of 37 genes. The arrangement of these genes and the major noncoding region is relatively conserved through evolution although gene rearrangements have been described for diverse lineages. The tandem duplication-random loss model has been invoked to explain the mechanisms of most mitochondrial gene rearrangements. Previously reported mitogenomic sequences for geckos rarely included gene rearrangements, which we explore in the present study.

Results

We determined seven new mitogenomic sequences from Gekkonidae using a high-throughput sequencing method. The Tropiocolotes tripolitanus mitogenome involves a tandem duplication of the gene block: tRNA^Arg, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 4. One of the duplicate copies for each protein-coding gene may be pseudogenized. A duplicate copy of the tRNA^Arg gene appears to have been converted to a tRNA^Gln gene by a C to T base substitution at the second anticodon position, although this gene may not be fully functional in protein synthesis. The Stenodactylus petrii mitogenome includes several tandem duplications of tRNA^Leu genes, as well as a translocation of the tRNA^Ala gene and a putative origin of light-strand replication within a tRNA gene cluster. Finally, the Uroplatus fimbriatus and U. ebenaui mitogenomes feature the apparent loss of the tRNA^Glu gene from its original position. Uroplatus fimbriatus appears to retain a translocated tRNA^Glu gene adjacent to the 5’ end of the major noncoding region.

Conclusions

The present study describes several new mitochondrial gene rearrangements from Gekkonidae. The loss and reassignment of tRNA genes is not very common in vertebrate mitogenomes and our findings raise new questions as to how missing tRNAs are supplied and if the reassigned tRNA gene is fully functional. These new examples of mitochondrial gene rearrangements in geckos should broaden our understanding of the evolution of mitochondrial gene arrangements.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-930) contains supplementary material, which is available to authorized users.     

The complete mitochondrial genomes of sixteen ardeid birds revealing the evolutionary process of the gene rearrangements

Background

The animal mitochondrial genome is generally considered to be under selection for both compactness and gene order conservation. As more mitochondrial genomes are sequenced, mitochondrial duplications and gene rearrangements have been frequently identified among diverse animal groups. Although several mechanisms of gene rearrangement have been proposed thus far, more observational evidence from major taxa is needed to validate specific mechanisms. In the current study, the complete mitochondrial DNA of sixteen bird species from the family Ardeidae was sequenced and the evolution of mitochondrial gene rearrangements was investigated. The mitochondrial genomes were then used to review the phylogenies of these ardeid birds.

Results

The complete mitochondrial genome sequences of the sixteen ardeid birds exhibited four distinct mitochondrial gene orders in which two of them, named as “duplicate tRNA^Glu–CR” and “duplicate tRNA^Thr–tRNA^Pro and CR”, were newly discovered. These gene rearrangements arose from an evolutionary process consistent with the tandem duplication - random loss model (TDRL). Additionally, duplications in these gene orders were near identical in nucleotide sequences within each individual, suggesting that they evolved in concert. Phylogenetic analyses of the sixteen ardeid species supported the idea that Ardea ibis, Ardea modesta and Ardea intermedia should be classified as genus Ardea, and Ixobrychus flavicollis as genus Ixobrychus, and indicated that within the subfamily Ardeinae, Nycticorax nycticorax is closely related to genus Egretta and that Ardeola bacchus and Butorides striatus are closely related to the genus Ardea.

Conclusions

The duplicate tRNAThr–CR gene order is found in most ardeid lineages, suggesting this gene order is the ancestral pattern within these birds and persisted in most lineages via concerted evolution. In two independent lineages, when the concerted evolution stopped in some subsections due to the accumulation of numerous substitutions and deletions, the duplicate tRNAThr–CR gene order was transformed into three other gene orders. The phylogenetic trees produced from concatenated rRNA and protein coding genes have high support values in most nodes, indicating that the mitochondrial genome sequences are promising markers for resolving the phylogenetic issues of ardeid birds when more taxa are added.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-573) contains supplementary material, which is available to authorized users.     

The hydrolysis of primary amide groups in Asn/Gln-containing peptides

Peptides Boc-Ala-Asn/Gln-OH and Boc-Asn/Gln-Ala-OH were saponified with barium hydroxide to corresponding Asp/Glu-containing peptides. Under the conditions of saponification, Boc-Asn-Ala-OH additionally afforded Boc-Asp-OH, isopeptide Boc-Asp(Ala)-OH, and Boc-NHSuc>Ala-OH, with the third being the key intermediate in these transformations. Boc-Asp(OMe)-Ala-OMe underwent similar transformations under treatment with diazomethane or triethylamine. Saponification with barium hydroxide was accompanied by a high epimerization ofN-terminal amino acid residues, whereas the products of the diazomethane treatment of Boc-Asp(OMe)-Ala-OMe had a low degree of epimerization.     

Meiosis and the Neo- XY system of Dichroplus vittatus (Melanoplinae, Acrididae): a comparison between sexes

The origin of neo-XY sex systems in Acrididae is usually explained through an X-autosome centric fusion, and the behaviour of the neo-sex chromosomes has been solely studied in males. In this paper we analysed male and female Dichroplus vittatus. The karyotype comprises 2n = 20 chromosomes including 9 pairs of autosomes and a sex chromosome pair that includes a large metacentric neo-X and a small telocentric neo-Y. We compared the meiotic behaviour of the sex bivalent between both sexes. Mean cell autosomal chiasma frequency was low in both sexes and slightly but significantly higher in males than in females. Chiasma frequency of females increased significantly when the sex-bivalent was included. Chiasma distribution was basically distal in both sexes. Behaviour of the neo-XY pair is complex as a priori suggested by its structure, which was analysed in mitosis and meiosis of diploid and polyploid cells. During meiosis, orientation of the neo-XY is highly irregular; only 21% of the metaphase I spermatocytes show standard orientation. In the rest of cells, the alternate or simultaneous activity of an extra kinetochore in the distal end of the short arm (XL) of the neo-X, determined unusual MI orientations and a high frequency of non-disjunction and lagging of the sex-chromosomes. In females, the neo-XX bivalent had a more regular behaviour but showed 17% asynapsis in the XL arm which, in those cases orientated its distal ends towards opposite spindle poles suggesting, again, the activity of a second kinetochore. The dicentric nature and the unstable meiotic behaviour of the sex neo-chromosomes of D. vittatus suggest a recent origin of the sex determination mechanism, with presumable adaptive advantages which could compensate their potential negative heterosis. Our observations suggest that the origin of the neo-sex system was a tandem fusion of two original telocentric X-chromosomes followed by another tandem fusion with the small megameric bivalent and a further pericentric inversion of the neo-X. The remaining autosomal homolog resulted in the neo-Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.