Microsatellites, or simple sequence repeats (SSRs) are very useful molecular markers for a number of plant species. They are commonly used in cultivar identification, plant variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Early development of SSRs was hampered by the high cost of library screening and clone sequencing. Currently, large public SSR datasets exist for many crop species, but the number of publicly available, mapped SSRs for potato is relatively low (approximately 100). We have utilized a database mining approach to identify SSR-containing sequences in The Institute For Genomic Research Potato Gene Index database (http://www.tigr.org), focusing on sequences with size polymorphisms present in this dataset. Ninety-four primer pairs flanking SSR sequences were synthesized and used to amplify potato DNA. This study rendered 61 useful SSRs that were located in pre-existing genetic maps, fingerprinted in a set of 30 cultivars from South America, North America, and Europe or a combination thereof. The high proportion of success (65%) of expressed sequence tag-derived SSRs obtained in this work validates the use of transcribed sequences as a source of markers. These markers will be useful for genetic mapping, taxonomic studies, marker-assisted selection, and cultivar identification.

The Krü ppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krü ppel-like factor (EKLF or KLF1) positively regulates the ␤-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2 ؊/؊ mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the ␤-like globin genes in KLF2 knockout mice. Our results show that KLF2 ؊/؊ mice have a significant reduction of murine embryonic Ey-and ␤h1-globin but not -globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult ␤ maj -and ␤ min -globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic ⑀-globin gene but not the adult ␤-globin gene, suggesting that this developmentalstage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2 ؊ IntroductionHematopoiesis represents a complex differentiation pathway involving many transcription factors and growth factors that interact in a concerted fashion during mammalian development. 1 Transcription factors often exist as multigene families of structurally and functionally related members that are involved in the different stages of development of a particular cell lineage. For example, members of the GATA family of transcription factors are involved in both primitive and definitive erythropoiesis. 2-4 Different phenotypic features seen after ablation of either the GATA1 or the GATA2 gene in mice clearly demonstrate that these factors have different but overlapping functions. The Krüppel-like factors (KLFs) are a family of DNA-binding proteins named after the Drosophila Krüppel protein. KLFs have 3 C2/H2 zinc finger domains and share conserved residues located primarily within these zinc fingers. 5,6 Several of the KLFs are expressed in erythroid cells starting early in development. Erythroid Krüppel-like factor (EKLF or KLF1) was the first of 16 KLFs to be identified. EKLF Ϫ/Ϫ mice develop fatal anemia during fetal liver erythropoiesis. 7,8 EKLF is responsible for positively regulating the adult ␤-globin gene, but it is not required for embryonic/fetal globin gene expression. [9][10][11] Other KLF family members may be involved in the developmental control of the embryonic and fetal globin genes. A few of the KLFs, namely KLF2 and KLF5, 12 and KLF11 and KLF13,13,14 have been shown to activate the fetal ␥-globin gene in transient transfection assays in human erythroleukemia cell lines. So far none of these studies have been replicated in vivo. In a recent study, KLF11 (fetal Krüppel like factor, FKLF1)-null mice were found to be fertile, with normal hematopoiesis at all stages of development. There was no effe...

The developmental regulation of the human globin genes involves a key switch from fetal (y-) to adult (13-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the 13-globin locus. High-level, correct developmental regulation of 1-globin gene expression in transgenic mice has previously been accomplished only in 30-to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the 13-globin locus is required to silence the 13-globin gene in embryonic life. The results presented here clearly show that the presence of the 'y-globin gene (3.3 kb) alone is sufficient to down-regulate the 1,-globin gene in embryonic transgenic mice made with an LCR--1B-globin mini construct.The results also show that the y-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-y-globin constructs not including the 13-globin gene, i.e., that the y-tglobin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the -y-globin gene may be important for down-regulation in the adult. The 5'HS2yenl3 construct described is a suitable model for further study of the mechanism of human y-to 13-globin gene switching in transgenic mice.

The genes of the vertebrate ␤-globin locus undergo a switch in expression during erythroid development whereby embryonic͞ fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal ␥-globin gene. The ␥-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human ␤-globin locus as a yeast artificial chromosome (␤YAC) transgene. To elucidate the mechanism through which DNA methylation represses the ␥-globin gene in adult erythroid cells, ␤YAC͞MBD2؊/؊ mice were generated by breeding ␤YAC mice with MBD2؊͞؊ mice. Adult ␤YAC͞MBD2؊/؊ mice continue to express the ␥-globin gene at a level commensurate with 5-azacytidine treatment, 10-to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of ␥-globin expression is consistently higher in MBD2؊͞؊ mice in 14.5-and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic͞fetal erythroid development. DNA methylation levels are modestly decreased in MBD2؊͞؊ mice. MBD2 does not bind to the ␥-globin promoter region to maintain ␥-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of ␥-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain ␥-globin suppression in adult erythroid cells.DNA methylation ͉ epigenetics ͉ transcription

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BackgroundKrüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.ResultsEndothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.ConclusionsThis study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.

Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.

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