Osmium tetroxide footprinting of a scaffold attachment region in the maizeAdh1 promoter

Osmium tetroxide (OsO₄) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO₄ hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO₄ will also identify other torsional perturbations of DNA. In this study, OsO₄ was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO₄ hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.     

Effects of promoter,intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA -glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5 transcribed region decreased expression strength.     

Wheat acetyl-CoA carboxylase

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of ¹⁴C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.     

DNA binding factor GT-2 from Arabidopsis

Complementary DNA clones encoding a DNA-binding factor have been obtained from Arabidopsis by DNA hybridization with a GT-2 factor cDNA clone from rice. The GT-2 gene appears to be present as a single copy in the Arabidopsis genome and is transcribed as a 2.1 kb mRNA which is not light-regulated. The longest open reading frame in the sequenced clones predicts a protein of 65 kDa, beginning with the first in-frame methionine. The protein contains basic, acidic, and proline/glutamine-rich motifs and has significant amino acid sequence homology to the rice GT-2 factor, including three regions of 50–75 amino acids each of greater than 60% identity. Two of these regions are predicted to form similar trihelix structures postulated to be involved in selective binding to specific variations of a GT-box motif DNA sequence found in the promoter regions of several plant genes. Except for weak similarity to a tobacco GT-box binding factor, GT-1a/B2F, Arabidopsis GT-2 has no similarity to other sequences in the databases. DNA-binding studies show that Arabidopsis GT-2 has binding characteristics similar to those of the rice GT-2 factor, but dissimilar to those of the tobacco GT-1a/B2F factor. The data indicate that a DNA-binding factor containing domains of similar structure and target-sequence specificity has been conserved between monocots and dicots.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.     

Songs of American Redstarts (Setophaga ruticilla): Sequencing Rules and their Relationships to Repertoire Size

In order to determine the rules of sequencing of songs used by American redstarts, we related Markovian and hierarchical models to recordings obtained from free-living males. In the smaller repertoires of three or four songs, low order Markov chain models fitted the data. 9 of the 10 sequences so examined were first-order, and the last was second-order. Larger repertoires of 6 and 8 songs were hierarchical in organization with subsets of songs having independent sequencing rules. Most samples of singing were stationary in their transition rules over periods of several days: non-stationarity was sometimes associated with a change in the number of songs forming the sequence, or in repetitions of songs. We examine causal models of song sequencing and conclude that our results generally favor competition models, although some sequential dependencies may also apply. Hierarchical organization in the serial repertoires of American redstarts may reflect developmental influences rather than effects of repertoire size itself.     

A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M_r, 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.     

Transport and transformation of hexavalent chromium through soils and into ground water

A detailed characterization of the underlying and adjacent soils of a chrome‐plating shop was performed to provide information on the extent of soil and aquifer contamination at the site and on the potential for off‐site migration and environmental impact. Intact, moist cores were obtained from more than 40 different locations, resulting in more than 200 discrete samples for total metal analysis, selective extraction tests, and adsorption‐reduction experiments, to assess the chemical speciation and distribution of chromium on the contaminated soils and its leaching potential. Surface analytical techniques were also used to determine chemical speciation and to further elucidate mineral fractions responsible for retention of the chromium on the soils and sediments. Adsorption and reduction capacities of the saturated aquifer sediments were variable and low, while the unsaturated soils’ reduction capacities were much greater and were correlated with depth (decreasing capacity with increasing depth). The soils’ adsorption and reduction capacities were eventually overwhelmed, however, and permitted the passage of Cr(VI) into the underlying ground water. Adsorption capacity differences were primarily related to clay content and pH, and less so to the presence of amorphous iron oxide coatings on matrix minerals as operationally defined by the selective extraction methods used in the study. Reduction of Cr(VI) to Cr(III) and subsequent precipitation as (Fe, Cr)(OH)₃ is proposed as the primary attenuation mechanism in the unsaturated soils immediately beneath the shop, based on extraction and surface analyses results.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.