Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs

Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked beta-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036-1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.     

Distinct geographic patterns of genetic diversity are maintained in wild barley (Hordeum vulgare ssp. spontaneum) despite migration

Mutations arise in a single individual and at a single point in time and space. The geographic distribution of mutations reflects both historical population size and frequency of migration. We employ coalescence-based methods to coestimate effective population size, frequency of migration, and level of recombination compatible with observed genealogical relationships in sequence data from nine nuclear genes in wild barley (Hordeum vulgare ssp. spontaneum), a highly self-fertilizing grass species. In self-fertilizing plants, gamete dispersal is severely limited; dissemination occurs primarily through seed dispersal. Also, heterozygosity is greatly reduced, which renders recombination less effective at randomizing genetic variation and causes larger portions of the genome to trace a similar history. Despite these predicted effects of this mating system, the majority of loci show evidence of recombination. Levels of nucleotide variation and the patterns of geographic distribution of mutations in wild barley are highly heterogeneous across loci. Two of the nine sampled loci maintain highly diverged, geographic region-specific suites of mutations. Two additional loci include region-specific haplotypes with a much shallower coalescence. Despite inbreeding, sessile growth habit, and the observation of geographic structure at almost half of sampled loci, parametric estimates of migration suggest that seed dispersal is sufficient for migration across the approximately 3,500-km range of the species. Recurrent migration is also evident based on the geographic distribution of mutational variation at some loci. At one locus a single haplotype has spread rapidly enough to occur, unmodified by mutation, across the range of the species.     

Restriction fragment length polymorphism-mediated targeting of the ml-o resistance locus in barley (Hordeum vulgare)

The ml-o locus in barley confers resistance to all known races of the fungus Erysiphe graminis f.sp. hordei. Since the molecular mechanisms underlying ml-o-mediated resistance are currently undefined, experiments have been initiated to isolate the gene by means of its map position. A collection of backcross lines containing ml-o alleles derived from six barley genotypes allowed us to identify a set of DNA markers very tightly linked to the resistance locus. These markers span an unexpectedly small segment of 8.6 centimorgans on chromosome 4 that includes the Ml-o locus. Two of the markers cosegregate with the resistance locus on the basis of 44 homozygous resistant plants identified within a segregating F2 population derived from an intravarietal cross. Colinearity of the resistance-linked markers was confirmed in an F2 mapping population derived from a wide cross between Hordeum vulgare subsp. vulgare and Hordeum vulgare subsp. spontaneum. The two markers cosegregating with the resistance locus in the former cross define in the latter cross an interval of 2.4 centimorgans within which Ml-o is most probably situated. The set of linked markers opens up the possibility of carrying out a bidirectional chromosomal walk or jump to the gene.     

Genetic evidence for a second domestication of barley (Hordeum vulgare) east of the Fertile Crescent

Cereal agriculture originated with the domestication of barley and early forms of wheat in the Fertile Crescent. There has long been speculation that barley was domesticated more than once. We use differences in haplotype frequency among geographic regions at multiple loci to infer at least two domestications of barley; one within the Fertile Crescent and a second 1,500-3,000 km farther east. The Fertile Crescent domestication contributed the majority of diversity in European and American cultivars, whereas the second domestication contributed most of the diversity in barley from Central Asia to the Far East.     

Low levels of linkage disequilibrium in wild barley (Hordeum vulgare ssp. spontaneum) despite high rates of self-fertilization

High levels of inbreeding cause populations to become composed of homozygous, inbred lines. High levels of homozygosity limit the effectiveness of recombination, and therefore, retard the rate of decay of linkage (gametic phase) disequilibrium (LD) among mutations. Inbreeding and recombination interact to shape the expected pattern of LD. The actual extent of nucleotide sequence level LD within inbreeding species has only been studied in Arabidopsis, a weedy species whose global range has recently expanded. In the present study, we examine the levels of LD within and between 18 nuclear genes in 25 accessions from across the geographic range of wild barley, a species with a selfing rate of approximately 98%. In addition to examination of intralocus LD, we employ a resampling method to determine whether interlocus LD exceeds expectations. We demonstrate that, for the majority of wild barley loci, intralocus LD decays rapidly, i.e., at a rate similar to that observed in the outcrossing species, Zea mays (maize). Excess interlocus LD is observed at 15% of two-locus combinations; almost all interlocus LD involves loci with significant geographic structuring of mutational variation.     

Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines

Aims/hypothesis  

Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim of the present study was to examine the KDAC gene expression profile of the beta cell and to investigate whether KDAC expression is regulated by cytokines. In addition, the protective effect of the non-selective KDAC inhibitor ITF2357 and interdependent regulation of four selected KDACs were investigated.     

Evolutionary Changes in the Mating System of an Experimental Population of Barley (Hordeum vulgare L.)

An analysis is presented of the reproductive cycle in an experimental population of barley. The experimental design included growing three different generations of the population in the same year and environment, thus permitting an assessment of the mating system at three stages in the evolutionary history of the population, unconfounded by environmental differences. Gene frequencies sometimes differed significantly in adults and in the effective pollen pool. It was also found that out-crossing rate more than doubled during the twenty generations spanned by the study. These results provide evidence for selection during reproductive phases of the life cycle. They also demonstrate that evolution in this predominantly self-pollinating population was in the direction of increased recombinational potential.     

Heterogeneous geographic patterns of nucleotide sequence diversity between two alcohol dehydrogenase genes in wild barley (Hordeum vulgare subspecies spontaneum)

Patterns of nucleotide sequence diversity in the predominantly self-fertilizing species Hordeum vulgare subspecies spontaneum (wild barley) are compared between the putative alcohol dehydrogenase 3 locus (denoted "adh3") and alcohol dehydrogenase 1 (adh1), two related but unlinked loci. The data consist of a sequence sample of 1,873 bp of "adh3" drawn from 25 accessions that span the species range. There were 104 polymorphic sites in the sequenced region of "adh3." The data reveal a strong geographic pattern of diversity at "adh3" despite geographic uniformity at adh1. Moreover, levels of nucleotide sequence diversity differ by nearly an order of magnitude between the two loci. Genealogical analysis resolved two distinct clusters of "adh3" alleles (dimorphic sequence types) that coalesce roughly 3 million years ago. One type consists of accessions from the Middle East, and the other consists of accessions predominantly from the Near East. The two "adh3" sequence types are characterized by a high level of differentiation between clusters ( approximately 2.2%), which induces an overall excess of intermediate frequency variants in the pooled sample. Finally, there is evidence of intralocus recombination in the "adh3" data, despite the high level of self-fertilization characteristic of wild barley.     

Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals

An important effector of Ca2+ signaling in animals and yeast is the Ca2+/calmodulin-dependent protein phosphatase calcineurin. However, the biochemical identity of plant calcineurin remained elusive. Here we report the molecular characterization of AtCBL (Arabidopsis thaliana calcineurin B-like protein) from Arabidopsis. The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase. AtCBL also shows significant similarity with another Ca2+-binding protein, the neuronal calcium sensor in animals. It contains typical EF-hand motifs with Ca2+-binding capability, as confirmed by in vitro Ca2+-binding assays, and it interacts in vivo with rat calcineurin A in the yeast two-hybrid system. Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant. Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in Arabidopsis. Genes for three isoforms were identified in this study. AtCBL1 mRNA was preferentially expressed in stems and roots and its mRNA levels strongly increased in response to specific stress signals such as drought, cold, and wounding. In contrast, AtCBL2 and AtCBL3 are constitutively expressed under all conditions investigated. Our data suggest that AtCBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions.     

Krebs cycle enzymes from livers of old mice are differentially regulated by caloric restriction

Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and alpha-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The alpha-ketoglutarate/glutamate and malate/alpha-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain.     

Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.

Two clones from a lambda phage collection containing yeast genes regulated by inorganic phosphate were shown by low-stringency hybridization to select three mRNAs that direct the in vitro synthesis of repressible acid phosphatase (EC 3.1.3.2) polypeptides p60, p58, and p56. By higher stringency hybridization one yeast fragment [8 kilobases (kb)] selects p60 mRNA and the other (5 kb) selects p56 mRNA. These EcoRI digestion fragments were subcloned in yeast transformation vectors and hybridization selection assignments were confirmed by measuring enzyme and mRNA levels in transformants. Enzyme and mRNA levels in (8-kb) high copy number transformants grown in high inorganic phosphate medium revealed a hitherto undetected acid phosphatase protein, P57, which is believed to correspond to the constitutive enzyme encoded by PHO3. The identify of the 8-kb fragment purported to contain the PHO5/PHO3 genes was confirmed by genetic mapping of an integrated copy of this fragment. The site of integration of the 5-kb fragment was demonstrated to be unlinked to the PHO5/PHO3 genes.     

Identification of four human cDNAs that are differentially expressed by early hematopoietic progenitors

The molecular processes that maintain the stem cell pool are largely unknown. Using polymerase chain reaction-driven subtraction, we examined genes that are differentially expressed by early hematopoietic progenitors. We expected that identifying genes that are uniquely expressed by the earliest precursors would provide insight into the mechanism(s) through which stem cell number is maintained and differentiation is regulated.Using CD34(+)CD38(-) cells as starting material, we identified four mRNAs, expressed by these cells, that are either absent or present in reduced amounts in more mature CD34(+)CD38(+) cells. One of these cDNAs (C40) encodes a known member of the subfamily of protein phosphatases (CL100) that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates and specifically inactivates MAP kinases. This phosphatase has been shown to play a role in regulating the differentiation of several cell types. The second cDNA (C23) is identical to LR11 (gp250), a member of the low-density lipoprotein receptor family. LR11 is unusual in that, in addition to 11 ligand-binding repeats, it contains a series of fibronectin type III repeats near its carboxyl terminal end that are similar to those found in cytokine receptors. It is highly expressed in developing brain, but hematopoietic expression has not been reported. The 178-bp fragment that we originally cloned is part of a 4,145-bp 3' untranslated region (UTR) that had not been previously sequenced and is among the largest human 3' UTRs ever reported. The other isolates (C21 and C12) do not correspond to known protein sequences. They are homologous to EST sequences from a fetal brain library. C21 encodes a previously unknown gene that is a member of the WD-40 family. An open reading frame encoding a 515 amino acid protein has been identified.Four mRNAs, differentially expressed by CD34(+)CD38(-) human bone marrow cells, have been identified. Although this population is highly enriched for early hematopoietic progenitors, none of these genes encodes a message whose expression is limited to the hematopoietic system. They all are expressed in a variety of tissues, suggesting that they are involved in processes that are fundamental to the development of many cell types. All of these cDNAs possess atypically long 3' UTRs, and one of them is among the longest ever described. Their differential expression by immature hematopoietic cells, in contrast to more mature cells, suggest that long 3' UTRs may be characteristic of genes that play a regulatory role during development.     

Differentiation and proliferation of periosteal osteoblast progenitors are differentially regulated by estrogens and intermittent parathyroid hormone administration

The periosteum is now widely recognized as a homeostatic and therapeutic target for actions of sex steroids and intermittent PTH administration. The mechanisms by which estrogens suppress but PTH promotes periosteal expansion are not known. In this report, we show that intermittent PTH(1-34) promotes differentiation of periosteal osteoblast precursors as evidenced by the stimulation of the expression or activity of alkaline phosphatase as well as of targets of the bone morphogenetic protein 2 (BMP-2) and Wnt pathways. In contrast, 17beta-estradiol (E2) had no effect by itself. However, it attenuated PTH- or BMP-2-induced differentiation of primary periosteal osteoblast progenitors. Administration of intermittent PTH to ovariectomized mice induced rapid phosphorylation of the BMP-2 target Smad1/5/8 in the periosteum. A replacement dose of E2 had no effect by itself but suppressed PTH-induced phosphorylation of Smad1/5/8. In contrast to its effects to stimulate periosteal osteoblast differentiation, PTH promoted and subsequently suppressed proliferation of periosteal osteoblast progenitors in vitro and in vivo. E2 promoted proliferation and attenuated the antiproliferative effect of PTH. Both hormones protected periosteal osteoblasts from apoptosis induced by various proapoptotic agents. These observations suggest that the different effects of PTH and estrogens on the periosteum result from opposing actions on the recruitment of early periosteal osteoblast progenitors. Intermittent PTH promotes osteoblast differentiation from periosteum-derived mesenchymal progenitors through ERK-, BMP-, and Wnt-dependent signaling pathways. Estrogens promote proliferation of early osteoblast progenitors but inhibit their differentiation by osteogenic agents such as PTH or BMP-2.     

Glucose transport and antilipolysis are differentially regulated by the polar head group of an insulin-sensitive glycophospholipid.

A glycophospholipid has been purified from rat liver membranes, which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid, which is a phosphooligosaccharide, was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. There was an "insulin-like" inhibitory effect of this phosphooligosaccharide on isoproterenol-stimulated lipolysis in adipocytes, whereas there was no effect on glucose oxidation under conditions that measure glucose transport. The antilipolytic effect of this phosphooligosaccharide was demonstrated in intact adipocytes. There was a linear correlation between the concentration of phosphooligosaccharide and its antilipolytic effect, the magnitude and time course of which were similar to that obtained with physiological concentrations of insulin. Submaximal concentrations of insulin and phosphooligosaccharide produced an additive antilipolytic effect. The antilipolytic effect of the phosphooligosaccharide was demonstrated only after release of this compound from the precursor glycophospholipid with phosphatidylinositol-specific phospholipase C, and the activity of the phosphooligosaccharide was sensitive to alkali. It is proposed that this phosphooligosaccharide plays a role in mediating certain insulin actions.     

J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.

Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization.