High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -     

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.     

Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.     

Oxidation of phenolic and non-phenolic substrates by the lignin peroxidase of Streptomyces viridosporus T7A

Summary Numerous single-ring, aromatic, phenolic and non-phenolic compounds were tested as substrates of Streptomyces viridosporus T7A extracellular lignin peroxidase. Oxidations were monitored by spectroscopy, with and without 4-aminoantipyrine (4-AAP) as a color-forming reagent. The oxidation of phenols containing one or no carbon groups in the para position resulted in coupling with 4-AAP to form a red color. Thin layer chromatography and mass spectroscopy showed that the oxidation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) resulted in a direct coupling between 4-AAP and the phenol ring to form a quinone structure. In the reaction with vanillyl acetone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and 4-AAP, 4-AAP coupled to Á-carbon of vanillyl acetone. As shown by UV-visible spectroscopy, S. viridosporus T7A peroxidase oxidized phenolic compounds, but was unable to oxidize non-phenolic ones.Paper no. 91 517 of the Idaho Agricultural Experiment Station Correspondence to: D. L. Crawford     

Structural instability of a transgene locus in tobacco is associated with aneuploidy

This paper describes molecular and cytogenetic evidence for the instability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro , suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcino-genesis are discussed.