Cloning and characterization of the HpaII methylase gene.

The HpaII restriction-modification system from Haemophilus parainfluenzae recognizes the DNA sequence CCGG. The gene for the HpaII methylase has been cloned into E. coli and its nucleotide sequence has been determined. The DNA of the clones is fully protected against cleavage by the HpaII restriction enzyme in vitro, indicating that the methylase gene is active in E. coli. The clones were isolated in an McrA-strain of E. coli; attempts to isolate them in an McrA+ strain were unsuccessful. The clones do not express detectable HpaII restriction endonuclease activity, suggesting that either the endonuclease gene is not expressed well in E. coli, or that it is not present in its entirety in any of the clones that we have isolated. The derived amino acid sequence of the HpaII methylase shows overall similarity to other cytosine methylases. It bears a particularly close resemblance to the sequences of the HhaI, BsuFI and MspI methylases. When compared with three other methylases that recognize CCGG, the variable region of the HpaII methylase, which is believed to be responsible for sequence specific recognition, shows some similarity to the corresponding regions of the BsuFI and MspI methylases, but is rather dissimilar to that of the SPR methylase.     

Protein binding elements in the human beta-polymerase promoter.

The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein.     

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.     

Absorption, translocation, and metabolism of14C-clomazone in soybean (Glycine max) and threeAmaranthus weed species

The patterns of clomazone (2-[(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxazolidinone) absorption, translocation, and metabolism and their contribution to the plant selectivity of this herbicide were studied in tolerant soybean [Glycine max (L.) Merr.] andAmaranthus hybridus and in susceptibleA. retroflexus andA. lividus. Differential root absorption appeared to play a significant role in the differential response of these four plant species to clomazone. Absorption of root-applied¹⁴C-clomazone was greater by the two sensitiveAmaranthus weeds than by the tolerant soybean andA. hybri-dus. Following application of¹⁴C-clomazone to roots, most of the absorbed radioactivity was translocated to the leaves of all four species. Approximately 50% of the absorbed¹⁴C-clomazone was metabolized by all four plant species as early as 12 h after treatment. Thin layer Chromatographic (TLC) analysis of plant tissue extracts from all four species revealed the formation of two major metabolites of clomazone. These unidentified metabolites had Rf values of 0.4 and 0.8, respectively, in a butanolacetic acidwater (1235, vol/vol/vol) developing system. The Rf value of unaltered clomazone in this system was 0.95. Differential metabolism or differential rate of metabolism of clomazone was not observed in this study and did not seem to account for the tolerance of soybean andA. hybridus or the suceptibility ofA. retroflexus andA. lividus to this herbicide.Plant Pathology, Physiology, and Weed Science Department, Contribution No. 600.     

An immunocytochemical study of keratin reactivity during rat odontogenesis

Summary The cytokeratin distribution in the developing rat enamel organ from day 15 of gestation through to 11 days post partum was examined immunohistochemically using a panel of monoclonal antibodies. A temporo-spatial programme of keratin expression was observed during odontogenesis and positive reactivity of the enamel organ was seen with the pan keratin antibodies CK1 (clone LP34 — reacts with a number of keratins including 6 and 18) and AE1-3 (reacts with most acidic and basic keratins). No reactivity was observed in the enamel organ with the other antibodies examined (Ks 8.12 [reacts with keratins 13 and 16], Ks 8.60 [reacts with keratins 10 and 11) and MCA157 [reacts with rat liver antigen]), although these antibodies did stain other epithelial tissues. This study supports the view that the epithelial cells of the enamel organ synthesize a tissuspecific subset of keratins which are related to the differentiation of the cells.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Mutational analysis of the tobacco mosaic virus 5''-leader for altered ability to enhance translation.

Mutational analysis of the 5'-untranslated leader sequence (omega) of tobacco mosaic virus (TMV) was carried out to determine those sequences necessary for the translational enhancement associated with omega. Five deletion mutants, a single base substitution, and a 25 base replacement mutant were tested for alterations in omega's ability to enhance expression of beta-glucuronidase (GUS) mRNA in tobacco mesophyll protoplasts and Escherichia coli or chloramphenicol acetyltransferase (CAT) mRNA in Xenopus laevis oocytes. Alteration of an eight base subsequence required for the binding of a second ribosome resulted in the loss of translational enhancement in X. laevis oocytes but not in protoplasts. Substantial increases in enhancement were observed for several of the mutants in E. coli.     

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells.

Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.