In the liver, glucokinase (GCK) facilitates hepatic glucose uptake during hyperglycemia and is essential for the regulation of a network of glucose-responsive genes involved in glycolysis, glycogen synthesis, and lipogenesis. To better understand the consequences of changes in response to a liver-specific deficiency of GCK function we examined the expression profiles of genes involved in glucose metabolism in the liver, pancreas, muscle and adipose tissue in heterozygous liver-specific Gck knockout (Gckw/−) mice. Our results showed that with the development of a liver GCK deficiency, significant decreases in the mRNA levels for insulin receptor and Glut2 were observed in the liver, and HkII in muscle, while glucagon mRNA increased markedly in the pancreas. The levels of circulating glucagon hormone levels increased with increased mRNA levels. Depite a decrease in muscle HkII levels, the hexokinase activity level did not change. Our findings suggest that in liver-specific Gckw/− mice, peripheral tissues use different strategies to tackle with hyperglycemia even at a young age. By identifying the specific changes that occur in different tissues at an early stage of glucokinase deficiency, potentially we can develop interventions to prevent further progression to diabetes.
PMID: 23085254 CAMSID: cams2717
liver glucokinase; gene expression; diabetes
Bats and cetaceans (i.e., whales, dolphins, porpoises) are two kinds of mammals with unique locomotive styles and occupy novel niches. Bats are the only mammals capable of sustained flight in the sky, while cetaceans have returned to the aquatic environment and are specialized for swimming. Associated with these novel adaptations to their environment, various development changes have occurred to their body plans and associated structures. Given the importance of Hox genes in many aspects of embryonic development, we conducted an analysis of the coding regions of all Hox gene family members from bats (represented by Pteropus vampyrus, Pteropus alecto, Myotis lucifugus and Myotis davidii) and cetaceans (represented by Tursiops truncatus) for adaptive evolution using the available draft genome sequences. Differences in the selective pressures acting on many Hox genes in bats and cetaceans were found compared to other mammals. Positive selection, however, was not found to act on any of the Hox genes in the common ancestor of bats and only upon Hoxb9 in cetaceans. PCR amplification data from additional bat and cetacean species, and application of the branch-site test 2, showed that the Hoxb2 gene within bats had significant evidence of positive selection. Thus, our study, with genomic and newly sequenced Hox genes, identifies two candidate Hox genes that may be closely linked with developmental changes in bats and cetaceans, such as those associated with the pancreatic, neuronal, thymus shape and forelimb. In addition, the difference in our results from the genome-wide scan and newly sequenced data reveals that great care must be taken in interpreting results from draft genome data from a limited number of species, and deep genetic sampling of a particular clade is a powerful tool for generating complementary data to address this limitation.
To investigate the functional and structural renal changes in a long-term liver-specific glucokinase knockout mouse, model of MODY2. Hemizygous glucokinase knockout mice, gckw/− groups, were compared at 6-, 10- and 14-months to their age-matched normal littermates, gckw/w groups. To examine changes we compared body weight, fasting blood glucose, serum insulin and creatinine levels, as well as 24 h urine samples that were collected for urine volume and protein analysis between the two groups. Renal tissues were collected and stained with hemotoxylin-eosin and periodic-acid Schiff, for light microscopic observation. Expression of renal Transforming Growth Factor β1 (TGF-β1) was determined by Western blot. Our results show that fasting blood glucose levels were significantly higher in gckw/− mice compared to gckw/w mice (P<0.01) for all age groups. Compared to age-matched gckw/w mice, 10-month old gckw/− mice have significantly elevated body weights (P<0.01), and urine volume (P<0.05) and protein concentrations (P<0.01). Renal tubular casts were observed in 10- and 14-month gckw/− mice. Significant increase in mesangial matrix and thickening of the glomerular basement membrane was observed in gckw/− compared to age-matched gckw/w mice at 10 and 14 months. As mice age increases, the levels of renal TGF-β1 are observed in both gckw/− and gckw/w mice. Our results indicate that renal changes occur in the liver-specific glucokinase knockout mouse model of MODY2, and suggest that TGF-β1 may play a key role in pathogenesis of these renal changes.
PMID: 21316027 CAMSID: cams2938
MODY; Diabetic nephropathy; Animal models
Liver glucokinase (GCK) deficient mice possess mild renal complications associated with diabetes. To investigate the progression of kidney disease and identify candidate genes involved in the pathogenesis of renal damage, we examined changes in tissue structure and gene expression in the kidneys of liver-specific GCK knockout (gckw/−) mice and age-matched normal wild-type control (gckw/w) mice as they aged. Suppression subtractive hybridization (SSH) was used to identify candidate genes that showed a pattern of differential expression between kidneys of gckw/− and gckw/w mice at 60 weeks of age. Differential expression of the candidate genes was examined by real-time qPCR in liver-specific gckw/− and gckw/w mice at 16, 26, 40, 60, and 85 weeks of age. Among the candidate genes, only glutathione peroxidase-3 (GPX3) was confirmed to show differential expression by qPCR in the 60-week old mice, however two others genes, MALAT1 and KEG, showed significant changes at other ages. This study shows that liver-specific glucokinase deficient mice display changes in kidney morphology by 40 weeks of age, and that renal complication may be correlated with a reduction in GPX3 levels. Since decreased GPX3 mRNA expression was observed at 26 weeks, which is younger than the age when pathological changes can be seen in kidney biopsies, GPX3 may serve as an early marker for kidney damage.
glucokinase (GCK); MODY2; differentially expressed genes; kidney; Gpx3
Coat color in dog breeds is an excellent character for revealing the power of artificial selection, as it is extremely diverse and likely the result of recent domestication. Coat color is generated by melanocytes, which synthesize pheomelanin (a red or yellow pigment) or eumelanin (a black or brown pigment) through the pigment type-switching pathway, and is regulated by three genes in dogs: MC1R (melanocortin receptor 1), CBD103 (β-defensin 103), and ASIP (agouti-signaling protein precursor). The genotypes of these three gene loci in dog breeds are associated with coat color pattern. Here, we resequenced these three gene loci in two Kunming dog populations and analyzed these sequences using population genetic approaches to identify evolutionary patterns that have occurred at these loci during the recent domestication and breeding of the Kunming dog. The analysis showed that MC1R undergoes balancing selection in both Kunming dog populations, and that the Fst value for MC1R indicates significant genetic differentiation across the two populations. In contrast, similar results were not observed for CBD103 or ASIP. These results suggest that high heterozygosity and allelic differences at the MC1R locus may explain both the mixed color coat, of yellow and black, and the difference in coat colors in both Kunming dog populations.
Since their divergence from the terrestrial artiodactyls, cetaceans have fully adapted to an aquatic lifestyle, which represents one of the most dramatic transformations in mammalian evolutionary history. Numerous morphological and physiological characters of cetaceans have been acquired in response to this drastic habitat transition, such as thickened blubber, echolocation, and ability to hold their breath for a long period of time. However, knowledge about the molecular basis underlying these adaptations is still limited. The sequence of the genome of Tursiops truncates provides an opportunity for a comparative genomic analyses to examine the molecular adaptation of this species. Here, we constructed 11,838 high-quality orthologous gene alignments culled from the dolphin and four other terrestrial mammalian genomes and screened for positive selection occurring in the dolphin lineage. In total, 368 (3.1%) of the genes were identified as having undergone positive selection by the branch-site model. Functional characterization of these genes showed that they are significantly enriched in the categories of lipid transport and localization, ATPase activity, sense perception of sound, and muscle contraction, areas that are potentially related to cetacean adaptations. In contrast, we did not find a similar pattern in the cow, a closely related species. We resequenced some of the positively selected sites (PSSs), within the positively selected genes, and showed that most of our identified PSSs (50/52) could be replicated. The results from this study should have important implications for our understanding of cetacean evolution and their adaptations to the aquatic environment.
Tursiops truncates; aquatic adaptation; positive selection; branch-site model
Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer’s disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.
The molecular basis of the evolution of phenotypic characters is very complex and is poorly understood with few examples documenting the roles of multiple genes. Considering that a single gene cannot fully explain the convergence of phenotypic characters, we choose to study the convergent evolution of rod vision in two divergent bats from a network perspective. The Old World fruit bats (Pteropodidae) are non-echolocating and have binocular vision, whereas the sheath-tailed bats (Emballonuridae) are echolocating and have monocular vision; however, they both have relatively large eyes and rely more on rod vision to find food and navigate in the night. We found that the genes CRX, which plays an essential role in the differentiation of photoreceptor cells, SAG, which is involved in the desensitization of the photoactivated transduction cascade, and the photoreceptor gene RH, which is directly responsible for the perception of dim light, have undergone parallel sequence evolution in two divergent lineages of bats with larger eyes (Pteropodidae and Emballonuroidea). The multiple convergent events in the network of genes essential for rod vision is a rare phenomenon that illustrates the importance of investigating pathways and networks in the evolution of the molecular basis of phenotypic convergence.
The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA–seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes.
The origin of genes can involve mechanisms such as gene duplication, exon shuffling, retroposition, mobile elements, lateral gene transfer, gene fusion/fission, and de novo origination. However, de novo origin, which means genes originate from a non-coding DNA region, is considered to be a very rare occurrence. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee, supported by both transcriptional and proteomic evidence. It is inconsistent with the traditional view that the de novo origin of new genes is rare. RNA–seq data indicate that these de novo originated genes have their highest expression in the cerebral cortex and testes, suggesting these genes may contribute to phenotypic traits that are unique to humans, such as development of cognitive ability. Therefore, the importance of de novo origination needs greater appreciation.
Lysozyme c (chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known.
Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution.
The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.
Proteins involved in a pathway are likely to evolve in a correlated fashion, and coevolving gene families tend to undergo complementary gains and losses. Accordingly, gene copy numbers (i.e., repertoire size) tend to show parallel changes during the evolution of coevolving gene families. To test and verify this hypothesis, here we describe positive correlations among the repertoire sizes of six gene families, that is, trypsin-like serine protease, odorant-binding protein, odorant receptor, gustatory receptor, cytochrome P450, and glutathione S-transferase after excluding the possibility of phylogenetic constraint and random drift. The observed correlations are indicative of parallel changes in the repertoire sizes of the six gene families that are due to similar demands for the quantity of these different genes in different lineages of Drosophila. In conclusion, we propose that the correlated evolution among these six gene families in Drosophila is a signature of a parallel response to ecological adaptation.
correlated evolution; Drosophila; gene family
Rhodopsin, encoded by the gene Rhodopsin (RH1), is extremely sensitive to light, and is responsible for dim-light vision. Bats are nocturnal mammals that inhabit poor light environments. Megabats (Old-World fruit bats) generally have well-developed eyes, while microbats (insectivorous bats) have developed echolocation and in general their eyes were degraded, however, dramatic differences in the eyes, and their reliance on vision, exist in this group. In this study, we examined the rod opsin gene (RH1), and compared its evolution to that of two cone opsin genes (SWS1 and M/LWS). While phylogenetic reconstruction with the cone opsin genes SWS1 and M/LWS generated a species tree in accord with expectations, the RH1 gene tree united Pteropodidae (Old-World fruit bats) and Yangochiroptera, with very high bootstrap values, suggesting the possibility of convergent evolution. The hypothesis of convergent evolution was further supported when nonsynonymous sites or amino acid sequences were used to construct phylogenies. Reconstructed RH1 sequences at internal nodes of the bat species phylogeny showed that: (1) Old-World fruit bats share an amino acid change (S270G) with the tomb bat; (2) Miniopterus share two amino acid changes (V104I, M183L) with Rhinolophoidea; (3) the amino acid replacement I123V occurred independently on four branches, and the replacements L99M, L266V and I286V occurred each on two branches. The multiple parallel amino acid replacements that occurred in the evolution of bat RH1 suggest the possibility of multiple convergences of their ecological specialization (i.e., various photic environments) during adaptation for the nocturnal lifestyle, and suggest that further attention is needed on the study of the ecology and behavior of bats.
Correction to Wu DD, Irwin DM, Zhang YP: Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair. BMC Evol Biol 2008, 8:241.
Glucokinase (GCK) plays an important role in the regulation of carbohydrate metabolism. In the liver, phosphorylation of glucose to glucose-6-phosphate by GCK is the first step for both glycolysis and glycogen synthesis. However, some vertebrate species are deficient in GCK activity in the liver, despite containing GCK genes that appear to be compatible with function in their genomes. Glucokinase regulatory protein (GCKR) is the most important post-transcriptional regulator of GCK in the liver; it participates in the modulation of GCK activity and location depending upon changes in glucose levels. In experimental models, loss of GCKR has been shown to associate with reduced hepatic GCK protein levels and activity.
GCKR genes and GCKR-like sequences were identified in the genomes of all vertebrate species with available genome sequences. The coding sequences of GCKR and GCKR-like genes were identified and aligned; base changes likely to disrupt coding potential or splicing were also identified.
GCKR genes could not be found in the genomes of 9 vertebrate species, including all birds. In addition, in multiple mammalian genomes, whereas GCKR-like gene sequences could be identified, these genes could not predict a functional protein. Vertebrate species that were previously reported to be deficient in hepatic GCK activity were found to have deleted (birds and lizard) or mutated (mammals) GCKR genes. Our results suggest that mutation of the GCKR gene leads to hepatic GCK deficiency due to the loss of the stabilizing effect of GCKR.
Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.
Many conserved secondary structures have been identified within conserved elements in the human genome, but only a small fraction of them are known to be functional RNAs. The evolutionary variations of these conserved secondary structures in human populations and their biological functions have not been fully studied.
We searched for polymorphisms within conserved secondary structures and identified a number of SNPs within these elements even though they are highly conserved among species. The density of SNPs in conserved secondary structures is about 65% of that of their flanking, non-conserved, sequences. Classification of sites as stems or as loops/bulges revealed that the density of SNPs in stems is about 62% of that found in loops/bulges. Analysis of derived allele frequency data indicates that sites in stems are under stronger evolutionary constraint than sites in loops/bulges. Intergenic conserved secondary structures tend to associate with transcription factor-encoding genes with genetic distance being the measure of regulator-gene associations. A substantial fraction of intergenic conserved secondary structures overlap characterized binding sites for multiple transcription factors.
Strong purifying selection implies that secondary structures are probably important carriers of biological functions for conserved sequences. The overlap between intergenic conserved secondary structures and transcription factor binding sites further suggests that intergenic conserved secondary structures have essential roles in directing gene expression in transcriptional regulation networks.
Hair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and resistant hair shafts.
The KRTAP family was identified as being unique to mammals, and near-complete KRTAP gene repertoires for eight mammalian genomes were characterized in this study. An expanded KRTAP gene repertoire was found in rodents. Surprisingly, humans have a similar number of genes as other primates despite the relative hairlessness of humans. We identified several new subfamilies not previously reported in the high/ultrahigh cysteine KRTAP genes. Genes in many subfamilies of the high/ultrahigh cysteine KRTAP genes have evolved by concerted evolution with frequent gene conversion events, yielding a higher GC base content for these gene sequences. In contrast, the high glycine-tyrosine KRTAP genes have evolved more dynamically, with fewer gene conversion events and thus have a lower GC base content, possibly due to positive selection.
Most of the subfamilies emerged early in the evolution of mammals, thus we propose that the mammalian ancestor should have a diverse KRTAP gene repertoire. We propose that hair content characteristics have evolved and diverged rapidly among mammals because of rapid divergent evolution of KRTAPs between species. In contrast, subfamilies of KRTAP genes have been homogenized within each species due to concerted evolution.
For most mammals, running is their major locomotive style, however, cetaceans and bats are two mammalian groups that have independently developed new locomotive styles (swimming and flying) from their terrestrial ancestors. In this study, we used a genome-wide comparative analysis in an attempt to identify the selective imprint of the development of new locomotive styles by cetaceans and bats to adapt to their new ecological niches. We found that an elevated proportion of mitochondrion-associated genes show evidence of adaptive evolution in cetaceans and on the common ancestral lineage leading to bats, compared to other terrestrial mammals. This result is consistent with the fact that during the independent developments of swimming and flying in these two groups, the changes of energy metabolism ratios would be among the most important factors to overcome elevated energy demands. Furthermore, genes that show evidence of sequence convergence or parallel evolution in these two lineages were overrepresented in the categories of energy metabolism, muscle contraction, heart, and glucose metabolism, genes that perform functions which are essential for locomotion. In conclusion, our analyses showed that on the dolphin and bat lineages, genes associated with locomotion not only both show a greater propensity to adaptively evolve, but also show evidence of sequence convergence, which likely reflects a response to a common requirement during their development of these two drastic locomotive styles.
Glucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood.
To better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5′ pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5′ flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5′ flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3′ to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin.
Our results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.
The Tibetan antelope (Pantholops hodgsonii) is endemic to the extremely inhospitable high-altitude environment of the Qinghai-Tibetan Plateau, a region that has a low partial pressure of oxygen and high ultraviolet radiation. Here we generate a draft genome of this artiodactyl and use it to detect the potential genetic bases of highland adaptation. Compared with other plain-dwelling mammals, the genome of the Tibetan antelope shows signals of adaptive evolution and gene-family expansion in genes associated with energy metabolism and oxygen transmission. Both the highland American pika, and the Tibetan antelope have signals of positive selection for genes involved in DNA repair and the production of ATPase. Genes associated with hypoxia seem to have experienced convergent evolution. Thus, our study suggests that common genetic mechanisms might have been utilized to enable high-altitude adaptation.
The endemic Tibetan antelope is adapted to high-altitude environments with low partial pressure of oxygen and high level of ultraviolet radiation. Here Ge et al. report a draft genome of this species and by comparison with other mammals, present possible genetic bases of highland adaptation.