The divergence between monocots and dicots represents a major event in higher plant evolution, yet the date of its occurrence remains unknown because of the scarcity of relevant fossils. We have estimated this date by reconstructing phylogenetic trees from chloroplast DNA sequences, using two independent approaches: the rate of synonymous nucleotide substitution was calibrated from the divergence of maize, wheat, and rice, whereas the rate of nonsynonymous substitution was calibrated from the divergence of angiosperms and bryophytes. Both methods lead to an estimate of the monocotdicot divergence at 200 million years (Myr) ago (with an uncertainty of about 40 Myr). This estimate is also supported by analyses of the nuclear genes encoding large and small subunit ribosomal RNAs. These results imply that the angiosperm lineage emerged in Jurassic-Triassic time, which considerably predates its appearance in the fossil record (-120 Myr ago). We estimate the divergence between cycads and angiosperms to be -340 Myr, which can be taken as an upper bound for the age of angiosperms.The fossil record shows a vast increase in the numbers and distribution of angiosperm species in the mid-Cretaceous period, around 100 million years (Myr) ago (1). The earliest reliable angiosperm macrofossils are about 120 Myr old, but because these are already clearly divisible into monocotyledonous and dicotyledonous types it seems that the earliest stages of angiosperm evolution evaded fossilization (2, 3). Although it is generally accepted that angiosperms descended from the progymnosperm lineage, there is little agreement as to when they arose or even from which branch of the gymnosperms they stem (4). Since the progymnosperm lineage extends back to at least 370 Myr ago (2), there is an enormous range of time during which angiosperms might have had their beginnings. Theories as to why there are no fossils of progenitor angiosperms fall into two basic types: either angiosperms did not exist until the early Cretaceous and then radiated explosively, or pre-Cretaceous angiosperms lived in habitats so refractory to fossilization that they left no record (3-5). In this paper we attempt to decide between these alternative theories by analyzing plant DNA sequences, which can be used to estimate the date of divergence of monocots and dicots, and hence to provide a minimal age for angiosperms themselves.Despite promising early results from protein sequencing (for example, see ref. 6), molecular data have not been used extensively to investigate plant evolution. An initial application of DNA sequences to studying the origin of angiosperms has recently been made by Martin et al. (7). Their analysis is based on comparison between plants, animals, and fungi of the sequences of the nuclear gene (called gapC in plants) encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By using several divergence dates between animal taxa and between the plant, animal, and fungal kingdoms they were able to calculate the rate of evolution of this...

We obtained 16 nucleotide sequences ( approximately 1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16-0.23 x 10(-9) substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5-0.7 x 10(-9) substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170-235 million years (Myr) for the monocot-dicot split, 112-156 Myr for the Brassicaceae-Lettuce split, 14.5-20.4 Myr for the Brassica-Arabidopsis split, and 14.5-20.4 Myr for the Arabidopsis-Arabideae split.

Phylogenetic analysis of DNA sequences from primates, rodents, lagomorphs, artiodactyls, carnivores, and birds strongly suggests that the order Rodentia is an outgroup to the other four mammalian orders and that Artiodactyla and Carnivora belong to a superordinal clade. Further, there is strong evidence against the Glires concept, which unites Lagomorpha and Rodentia. The radiation among Lagomorpha,Primates, and Artiodactyla-Carnivora is very bush-like, but there is some evidence that Lagomorpha has branched off first. Thus, the branching sequence for these five orders of mammals seems to be Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora. The branching date for Rodentia could be as early as 100 million years ago. The rate of nucleotide substitution in the rodent lineage is shown to be at least 1.5 times higher than those in the other four mammalian lineages.Despite the efforts of numerous comparative anatomists, paleontologists, and molecular evolutionists (1-8), the branching order of the major eutherian lineages remains highly controversial. In fact, the prevailing view of eutherian evolution has always been a bush-like radiation (1, 2, 9). To untangle this phylogenetic knot, we have sequenced a number of apolipoprotein (Apo) genes from several mammalian orders and have compiled DNA sequences of these and other genes from data banks and the literature. Our current focus is on Primates, Rodentia, Lagomorpha, Artiodactyla, and Carnivora because there are many more DNA sequence data from these five orders than from others.

Glis3 is a member of the Krüppel-like family of transcription factors and is highly expressed in islet β cells. Mutations in GLIS3 cause the syndrome of neonatal diabetes and congenital hypothyroidism (NDH). Our aim was to examine the role of Glis3 in β cells, specifically with regard to regulation of insulin gene transcription. We demonstrate that insulin 2 (Ins2) mRNA expression in rat insulinoma 832/13 cells is markedly increased by wild-type Glis3 overexpression, but not by the NDH1 mutant. Furthermore, expression of both Ins1 and Ins2 mRNA is downregulated when Glis3 is knocked down by siRNA. Glis3 binds to the Ins2 promoter in the cell, detected by chromatin immunoprecipitation. Deletion analysis of Ins2 promoter identifies a sequence (5′-GTCCCCTGCTGTGAA-3′) from −255 to −241 as the Glis3 response element and binding occur specifically via the Glis3 zinc finger region as revealed by mobility shift assays. Moreover, Glis3 physically and functionally interacts with Pdx1, MafA and NeuroD1 to modulate Ins2 promoter activity. Glis3 also may indirectly affect insulin promoter activity through upregulation of MafA and downregulation of Nkx6-1. This study uncovers a role of Glis3 for regulation of insulin gene expression and expands our understanding of its role in the β cell.

To date, more than 30 genes have been linked to monogenic diabetes. Candidate gene and genome-wide association studies have identified > 50 susceptibility loci for common type 1 diabetes (T1D) and approximately 100 susceptibility loci for type 2 diabetes (T2D). About 1-5% of all cases of diabetes result from single-gene mutations and are called monogenic diabetes. Here, we review the pathophysiological basis of the role of monogenic diabetes genes that have also been found to be associated with common T1D and/or T2D. Variants of approximately one-third of monogenic diabetes genes are associated with T2D, but not T1D. Two of the T2D-associated monogenic diabetes genes-potassium inward-rectifying channel, subfamily J, member 11 (KCNJ11), which controls glucose-stimulated insulin secretion in the β-cell; and peroxisome proliferator-activated receptor γ (PPARG), which impacts multiple tissue targets in relation to inflammation and insulin sensitivity-have been developed as major antidiabetic drug targets. Another monogenic diabetes gene, the preproinsulin gene (INS), is unique in that INS mutations can cause hyperinsulinemia, hyperproinsulinemia, neonatal diabetes mellitus, one type of maturity-onset diabetes of the young (MODY10), and autoantibody-negative T1D. Dominant heterozygous INS mutations are the second most common cause of permanent neonatal diabetes. Moreover, INS gene variants are strongly associated with common T1D (type 1a), but inconsistently with T2D. Variants of the monogenic diabetes gene Gli-similar 3 (GLIS3) are associated with both T1D and T2D. GLIS3 is a key transcription factor in insulin production and β-cell differentiation during embryonic development, which perturbation forms the basis of monogenic diabetes as well as its association with T1D. GLIS3 is also required for compensatory β-cell proliferation in adults; impairment of this function predisposes to T2D. Thus, monogenic forms of diabetes are invaluable "human models" that have contributed to our understanding of the pathophysiological basis of common T1D and T2D.

Aims/hypothesis Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3−/− mice and to further decipher its action in in-vitro systems. Methods We created Glis3−/− mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components. Results In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2. Conclusions/interpretation These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice.

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Genome-wide association studies identified GLIS3 as a susceptibility locus for type 1 and type 2 diabetes. Global Glis3 deficiency in mice leads to congenital diabetes and neonatal lethality. In this study, we explore the role of Glis3 in adulthood using Glis3+/− and conditional knockout animals. We challenged Glis3+/− mice with high fat diet for 20 weeks and found that they developed diabetes because of impaired beta cell mass expansion. GLIS3 controls beta cell proliferation in response to high-fat feeding at least partly by regulating Ccnd2 transcription. To determine if sustained Glis3 expression is essential to normal beta cell function, we generated Glis3fl/fl/Pdx1CreERT+ animal by intercrossing Glis3fl/fl mice with Pdx1CreERT+ mice and used tamoxifen (TAM) to induce Glis3 deletion in adults. Adult Glis3fl/fl/Pdx1CreERT+ mice are euglycaemic. TAM-mediated beta cell-specific inactivation of Glis3 in adult mice downregulates insulin expression, leading to hyperglycaemia and subsequently enhanced beta cell apoptosis. We conclude that normal Glis3 expression is required for pancreatic beta cell function and mass maintenance during adulthood, which impairment leads to diabetes in adults.