Arabidopsis MAP-Kinase 3 Phosphorylates UDP-Glucose Dehydrogenase: a Key Enzyme Providing UDP-Sugar for Cell Wall Biosynthesis

The enzyme UDP-glucose dehydrogenase (UGD) competes with sucrose-phosphate synthase for the common photosynthesis product UDP-glucose. Sucrose-phosphate synthase is part of a pathway for the export of sucrose from source leaves to neighboring cells or the phloem. UGD is a central enzyme in a pathway for many nucleotide sugars used in local cell wall biosynthesis. Here, we identify a highly conserved phosphorylation site in UGD which is readily phosphorylated by MAP-kinase 3 in Arabidopsis. Phosphorylation occurs at a surface-exposed extra loop in all plant UGDs that is absent in UGDs from bacteria or animals. Phosphorylated sucrose-phosphate synthase is shifted to an inactive form which we did not measure for phosphorylated UGD. Plant UGDs have an extra loop which is phosphorylated by AtMPK3. Phosphorylation is not causing a reduction of UGD activity as found for the competitor enzymes and thus sets a preference for maintaining UDP-sugars at a constant level to prioritize cell wall biosynthesis.

     

Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching.

Switching of mating type in Saccharomyces cerevisiae is directional; MAT alpha cells recombine to transfer information from HMRa while MATa cells switch using the silent cassette at HML alpha. Genetic analysis recently has defined a 700 bp recombination enhancer approximately 29 kb from the left end of chromosome III that is necessary for directionality. The chromatin structure of this region differs strikingly in a- and alpha-cells. Mat alpha2p organizes a 3.7 kb chromatin domain that opposes interaction of trans-acting proteins with the enhancer. In a-cells lacking the alpha2 repressor, two footprinted regions flank an approximately 100 bp section having a unique DNA structure. This structural signature probably reflects interactions of proteins that result in directional mating type switching.     

Targeted expression of MYCN causes neuroblastoma in transgenic mice.

The proto-oncogene MYCN is often amplified in human neuroblastomas. The assumption that the amplification contributes to tumorigenesis has never been tested directly. We have created transgenic mice that overexpress MYCN in neuroectodermal cells and develop neuroblastoma. Analysis of tumors by comparative genomic hybridization revealed gains and losses of at least seven chromosomal regions, all of which are syntenic with comparable abnormalities detected in human neuroblastomas. In addition, we have shown that increases in MYCN dosage or deficiencies in either of the tumor suppressor genes NF1 or RB1 can augment tumorigenesis by the transgene. Our results provide direct evidence that MYCN can contribute to the genesis of neuroblastoma, suggest that the genetic events involved in the genesis of neuroblastoma can be tumorigenic in more than one chronological sequence, and offer a model for further study of the pathogenesis and therapy of neuroblastoma.     

A coalescent approach to the polymerase chain reaction.

A versatile algorithm is developed to model PCR on a computer. The method is based on a modification of the coalescent process and provides a general framework to analyse data from PCR. It allows for incorporation of the dynamics of the replication process as described in terms of the number of starting template molecules and cycle-dependent PCR efficiency. The simulation method generates, as a first step, the genealogy of a set of sequences sampled from a final PCR product. In a second step a mutation process is superimposed and the resulting data set is analysed. The efficiency of our algorithm enables us to get reliable approximations of various sample distributions. We demonstrate the relevance of our method with two applications: maximum likelihood estimation of the error rate in PCR and a test of homogeneity of the template.     

Deletions in Xq26.3–q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3–q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother’s white blood cells and 80–85% of the sister’s leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing ∼13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed ∼12 Mb of DNA around the FMR1 gene. Cytogenetic and molecular data revealed that ∼50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3–q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3–q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients. Received: 19 December 1996 / Accepted: 13 March 1997     

Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas

The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent.     

Expression inEscherichia coliof Y5-Mutant and N-terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions.