Study of interaction between NO radicals and Martin's spirosilane by means of IR spectroscopy.

The matrix isolation method is used to record the IR spectrum of C18H8O2F12Si in the 4000-500 cm(-1) range. To gain an IR spectrum with a sufficient resolution, this technique was used with neon as the dilution medium at 5 K. The generated species were characterized by in situ fourier transform infrared (FT-IR) spectroscopy. Once the Martin's spirosilane 1 (C18H8O2F12Si) was characterized, its reactivity toward NO was investigated under the same experimental conditions (i.e., using neon as a dilution medium at 5 K). In this case, the use of neon at very low temperature leads to the formation of a chemically inert matrix in which the species are trapped and isolated from one another, thus hindering consecutive reactions. As a consequence, intermediates can be observed. This approach allowed us to characterize the NO adduct, leading to the formation of 1-(NO). Concentration effects as well as annealing experiments were carried out. In addition to this experimental approach, products were identified by using reference spectra. Our results proved that, in the dilute phase, the reaction between 1 and NO radicals leads to the formation of an adduct. This stable species can further react with NO to form a more stable compound: 1-(NO)2. This proves the ability of such species to trap NO.

Development of a PCR-based diagnostic assay for the determination of KEL genotype in donor blood samples.

The polymorphism which determines expression of Kell and Cellano antigens on the redcell surface has been reported to be a single C-->T nucleotide substitution at residue 701 where T codes for the presence of Kell antigen. This was confirmed by the direct automated sequencing of PCR products amplified from individuals of known Kell phenotype. The substitution creates a Bsm I restriction enzyme site and this has formed the basis for the development of a PCR-based diagnostic assay for the determination of Kell phenotype in samples of donor blood. The assay is based on RFLP analysis of coamplified PCR products, one of which spans the K/k polymorphic site, and one control fragment which contains a Bsm I site. Digestion of the PCR products with Bsm I restriction enzyme and subsequent gel analysis of the digest allowed unequivocal determination of the K/k status of all of the samples tested.

The effect of histone H1 and DNA methylation on transcription.

We have previously shown that DNA methylation acts as a focus for the formation of inactive chromatin in vivo. We have investigated the mechanism further by in vitro transcription of a template containing two tRNA genes and an extensive (G+C)-rich sequence characteristic of a CpG island. The extent of transcription from the unmethylated or fully methylated template was assayed in the presence of varied levels of histone H1. The transcriptional activity of both templates was inhibited by increasing amounts of histone H1, although inhibition with the methylated template occurs at a lower H1:DNA ratio. The H1c variant shows the greatest preferential inhibition of the methylated template. We demonstrated that histone H1 complexed to DNA is one of the factors that inhibits transcription by preventing the formation of initiation complexes, particularly on methylated template, rather than the formation of disordered H1.DNA aggregates.

An intron-containing tRNAArg gene within a large cluster of human tRNA genes.

The insert within lambda Ht363, a recombinant selected from a bank of human genomic DNA cloned in lambda Ch4A, is described. Southern blot hybridization with a mixed tRNA[32P]pCp probe revealed the presence of four tRNA genes, which were shown to represent further copies of genes previously identified as a solitary tRNAGly gene and as a three gene cluster on two different recombinants. In vitro transcription of a fragment containing the three gene cluster revealed the presence of a further pol III gene, which was shown to be that for a tRNAArgTCT. This gene contains a 15 bp intron, the presence of which presumably prevented its detection on Southern blots by tRNA hybridisation. The gene is present in the previously reported cluster and occurs in higher copy number (> 7) in other arrangements in the genome. Most of the copies of the gene have related intron sequences.

Inactive chromatin spreads from a focus of methylation.

The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from limited MspI digestion. Our results show that inactive chromatin is present at unmethylated regions in partially methylated minichromosomes and can thereby inhibit gene expression. Spreading of the inactive chromatin is not inhibited by the presence of active promoters, nor is it a consequence of transcriptional inactivity.

The conformation of tRNA genes. Chemical modification studies.

It has been suggested that eukaryotic tRNA genes might adopt a higher order stem and loop structure to facilitate transcription by interaction of their variably spaced intragenic promoter blocks. Using sodium bisulphite, which reacts specifically with cytosine residues in single-stranded nucleic acids, no deamination of C in the TTCGAA sequence of the 3' ICR of a tRNA(Leu) gene could be detected under conditions which caused 60% deamination of cytosine residues within the loop region of a synthetic cruciform cloned in the same negatively supercoiled plasmid vector. We conclude that, under these conditions, such structures occur in tRNA genes very rarely, if at all.

Chromosomal assignment of a large tRNA gene cluster (tRNA(Leu), tRNA(Gln), tRNA(Lys), tRNA(Arg), tRNA(Gly)) to 17p13.1.

A cluster of tRNA genes (tRNA(UAGLeu), tRNA(CUGGln), tRNA(UUULys), tRNA(UCUArg)) and an adjacent tRNA(GCCGly) have been assigned to human chromosome 17p12-p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA(Leu), tRNA(Gln), tRNA(Lys), tRNA(Arg), and, for tRNA(Gly), 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100-p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotinylated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.

Human tRNAGlu genes: their copy number and organisation.

The tRNAGlu gene copy number, determined by genomic blot analysis of human placental DNA, is approximately thirteen. These studies, using several probes and DNA digested with several restriction enzymes singly or in combination, show that most of these tRNAGlu genes are flanked by DNA of very similar sequence for at least 5 kb. This conclusion is supported by the close similarity of the restriction maps of two lambda Charon-4A recombinants of human genomic DNA containing two different tRNAGlu genes.

The role of the 5'-flanking sequence of a human tRNA(Glu) gene in modulation of its transcriptional activity in vitro.

The role of a tRNA-like structure within the 5'-flanking sequence of a human tRNA(Glu) gene in the modulation of its transcription in vitro by HeLa cell extracts has been investigated using several deletion mutants of a recombinant of the gene which lacked part or all of the tRNA-like structure. The transcriptional efficiency of four mutants was the same as that of the wild-type recombinant, two mutants had decreased transcriptional efficiency, one was more efficient, and one, lacking part of the 5' intragenic control region, was inactive. Correlation of the transcriptional efficiencies with the position and the size of the 5'-flanking sequence that was deleted indicated that the tRNA-like structure may be deleted without loss of transcriptional efficiency. Current models for the modulation of tRNA gene transcription by the 5'-flanking sequence are assessed in the light of the results obtained, and a potential model is presented.

Chromosomal assignment of a glutamic acid transfer RNA (tRNAGlu) gene to 1p36.

A gene for tRNAGlu has been assigned to human chromosome 1p36 by in situ hybridisation using a [3H]-labelled or biotinylated 2.4-kb (human) DNA fragment containing a tRNAGlu gene as a probe. With the biotinylated DNA probe a secondary statistically significant site of hybridisation was observed at 1q21-22 which might represent a pseudogene or related sequence. In fibroblasts from gorilla (Gorilla gorilla) using biotin labelling, a single site of hybridisation occurred at 1qter which provides further support for homology of 1q in the higher apes and human 1p.

A human tRNAGlu gene of high transcriptional activity.

A mixture of low molecular weight RNAs, in which only tRNAs were radiolabelled, was used as a hybridisation probe to select for tRNA-like sequences within a bank of human genomic DNA in lambda Charon 4A. A restriction enzyme digest of one of the selected lambda Charon 4A recombinants contained two fragments (2.4 Kb & 1.8 Kb) which hybridised tRNA and which, when subcloned into pAT153, were transcribed in Xenopus oocyte nuclei. Analysis of the subcloned 2.4 Kb fragment, which was of remarkably high transcriptional activity, revealed the presence of a single gene for tRNAGlu in the middle of the fragment. The sequence immediately preceding the gene has the potential for forming a tRNA-like structure.