Studies of dotted,a regulatory element in maize

Summary Dotted (Dt) is the regulatory element of a two-unit controlling system in maize. Dt causes the inherited change from the recessive a ₁ (colorless) to its dominant allele, A ₁ (anthocyanin production), during the development of the stalk, leaves, and endosperm. The mutation events are observed as sectors of color in an anthocyaninless background.One of the most puzzling, but perhaps significant, aspects of controlling elements in maize is that they originate in conjunction with chromosome or chromatid breaks. This fact invokes a requirement that either an existing regulatory mechanism is disturbed by the breakage or that a foreign element is incorporated before fusion of the broken chromatids.Experimental crosses were made between Dt tester stocks and a pollen parent, a large proportion of whose chromosomes 9 were undergoing the chromatid type of bridge-breakage-fusion cycle. New Dt's were induced in endosperm sectors of 250 of 154,422 kernels tested (1/600); among these, two germinal Dt's (Dt ^crown ₄ and Dt ₅) were recovered, presumably due to chromatid breaks during meiosis or the first microspore division. Dt ₅ produces a mutation pattern very similar to the original Dt ₁ and is located 0.33 crossover units away from the yg ₂ locus. This is close to the known location of Dt ₁ (7 crossover units distal to the yg ₂ locus) and is suggestive of a specific site for Dt inductive breaks. Dt ^crown ₄, on the other hand, is inherited independently of the yg ₂ locus and does not support this contention. Dt ^crown ₄ represents a new state causing a high concentration of fine dots in the crown of the kernel, with little or no dotting at the base.The phase variation of Dt ^crown ₄ is discussed together with the tissue-dependent expression of Dt ^in-ac ₁ (Dotted, inactive-active). Dt ^in-ac ₁ is a new state of Dt ₁ and shows inactive (no a ₁ to A ₁ mutations) and active (a ₁ to A ₁ mutations) phases in the endosperm, whereas it is only in the active phase in the diploid scutellum. The observed phase variation was shown to be a property of the regulatory elements, Dt, responding to differences in the cellular environment.In appreciation of his help and guidance, I dedicate this article to Professor Marcus M. Rhoades on his birthday.Journal Paper No. J-7454 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1880.     

Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize

Summary Pseudomonas solanacearum, the causal agent of bacterial wilt, has been classified into three races based on host range and into five biovars based on physiological properties. Strains of race 3 belong exclusively to biovar 2 and primarily affect potatoes. Although this race is thought to have originated in the Andean highlands, it has unusual physiological properties that make it a potential threat to potatoes grown at the cooler latitudes worldwide. Consequently, there is need for a rapid and sensitive method for detection of race 3 strains. We have used subtractive hybridization to enrich for race 3-specific DNA sequences in total race 3 genomic DNA, and thereby obtained a 2 kb clone homologous to DNA from all 28 race 3 strains tested, but with only five of 90 non-race 3 strains. In addition, two larger regions of the genome, containing a minimum of 23 kb of DNA, are also specific for race 3. Deletion of this DNA did not affect virulence. This race 3-specific DNA is a potentially useful diagnostic tool for the detection of race 3 strains.     

Overexpression of the maize Teosinte Branched1 gene in wheat suppresses tiller development

The number of viable shoots influences the overall architecture and productivity of wheat (Triticum aestivum L.). The development of lateral branches, or tillers, largely determines the resultant canopy. Tillers develop from the outgrowth of axillary buds, which form in leaf axils at the crown of the plant. Tiller number can be reduced if axillary buds are not formed or if the outgrowth of these buds is restricted. The teosinte branched1 (tb1) gene in maize, and homologs in rice and Arabidopsis, genetically regulate vegetative branching. In maize, increased expression of the tb1 gene restricts the outgrowth of axillary buds into lateral branches. In this study, the maize tb1 gene was introduced through transformation into the wheat cultivar "Bobwhite" to determine the effect of tb1 overexpression on wheat shoot architecture. Examination of multiple generations of plants reveals that tb1 overexpression in wheat results in reduced tiller and spike number. In addition, the number of spikelets on the spike and leaf number were significantly greater in tb1-expressing plants, and the height of these plants was also reduced. These data reveal that the function of the tb1 gene and genetic regulation of lateral branching via the tb1 mode of action is conserved between wheat, rice, maize and Arabidopsis. Thus, the tb1 gene can be used to alter plant architecture in agriculturally important crops like wheat.     

R-stippled maize as a transposable element system

The I-R element at the R locus destabilizes kernel pigmentation giving the variegated pattern known as stippled ( R-st). In trans linkage phase with R-st the element was shown to act as a modifier of stippled, intensifying seed spotting in parallel with effects of the dominant linked modifier M-st. Presence of I-R in the genome was, therefore, shown to be detectable as a modifier of R-st. When this test was used, new modifiers resembling M-st were often detected following mutations of R-st to the stable allele R-sc. Such mutations evidently occurred by transposition of I-R away from the R locus to a site where it was identifiable as a modifier. M-st may be such a transposed I-R. Analysis of mutations to R-sc during the second (sperm-forming) mitosis in pollen grains showed that some of the transposed I-R elements were linked with R, whereas others assorted independently. Their strengths varied from barely discernible to a level equal to M-st. Overreplication frequently accompanied transposition at the sperm-forming mitosis, leading to transposed I-R elements in both the mutant and nonmutant sperm.     

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.     

An HLA-B regulatory element binds a factor immunologically related to the upstream stimulation factor

HLA-A and-B are expressed by most cell types, and their levels can be increased by treatment with interferons (IFNs). The relative basal levels of HLA-A and-B expression can vary, and HLA-B loci are induced much more strongly by IFNs. Constitutive activity is dependent on an upstream enhancer (ENH) which contains a rel (KBF, NFB) binding motif, and induction is mediated by an interferon response element (IRE) which binds members of the IRF family. Reported here is the identification of a regulatory element, R, which overlaps the IRE of HLA-B loci, but which is absent from the equivalent region of HLA-A or H2 class I genes. The core of the element, CACGAG, is bound by a nuclear factor which is recognized by an antiserum raised against the upstream stimulation factor (USF), a member of the helix-loop-helix/leucine zipper family. The use of reporter gene constructs shows that mutation of the R element results in increased induction by IFN in some cell lines, which appears to be due to competitive binding of USF with IRF proteins. Correspondence to: J. Girdlestone.     

Synaptic plasticity in an altered state

In this issue of Neuron, record from synaptically coupled pairs of CA3 neurons to closely examine the induction of synaptic depression at a small number of identified synapses. The authors provide convincing evidence that the activation history of a synapse determines both the ability of a synapse to depress and the mechanism of depression.     

crpX mutants of Escherichia coli K12: specific regulatory effects of altered cyclic AMP receptor proteins

Summary We attempted to correlate structural modifications of the adenosine 3,5 cyclic monophosphate (cAMP) receptor protein (CAP), to changes in some of its in vivo regulatory functions such as (i) stimulation of the lactose operon expression and (ii) control of adenylate cyclase activity. A radioimmunological procedure was used to study the structure of CAP synthesized by three mutants (crpX) grown under various conditions, in the presence or absence of endogenous or exogenous cAMP. In one mutant CAP appears to be sensitive to thermal inactivation. In another mutant CAP is particularly sensitive to degradation in the absence of cAMP; this degradation is enhanced by high temperature and during stationary phase of grwoth, and prevented by the addition of glucose. Functional alterations of CAP were not found to follow structural changes strictly. In the crpX mutants and in strains carrying the crp ⁺ or other crp allele, the stimulation of the lactose operon expression and the modulation of the in vivo rates of cAMP synthesis appear to vary in parallel, favoring an indirect mechanism of regulation of adenylate cyclase by CAP.     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Clustering of specific crossovers in maize microsporocytes

Analysis of systematic scan data from microsporocyte smear preparations suggests that cells with a crossover in a specific region tend to be geographically clustered to some extent within anthers. Crossover classes observed include single crossovers within a heterozygous inversion, three-strand double crossovers within and proximal to the inversion, and four-strand double crossovers within the inversion. Evidence for clustering within scans of cells of the first two classes is reported, but results are inconclusive for the third class. No evidence for within-scan correlation of frequencies of any two of the crossover classes was found. It is inferred that a large part of the variability in crossover frequency observed is probably due to factors which alter crossover interference. Implications related to the frequency and distribution of crossover sites are discussed.