John P. Goddard

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 structures and functions of transfer RNA.

Publisher Summary Transfer ribonucleic acid (tRNA) is the term used to describe a group of low molecular weight RNA molecules that play a vital part in protein synthesis Transfer RNAs were assigned the role of adaptor molecules by Hoagland et al. When, in the early 1960s, techniques for determining the nucleotide sequence of RNA molecules were developed, the tRNAs—the smallest species of RNA present in the cell—attracted further attention as relatively simple, naturally occurring nucleic acids that, it was hoped, would help in the elucidation of the general principles governing nucleic acid–protein interactions. The study of the biological role of tRNA in the past decade has not only given a fuller understanding of the mechanism of protein synthesis but has also shown that the role of tRNA in the cell is not confined to that of an adaptor molecule. However, attempts to relate the structure of tRNA even to its principal known function as an adaptor have met with mixed success. One of the major difficulties in relating tRNA structure to its function was that, until recently, the three-dimensional structure of tRNA was not known in detail. Physical and chemical studies of tRNAs in solution were limited to providing structural data that was either at a low resolution, giving only an indication of general shape, or resolved to the nucleotide level but restricted to selected regions of the molecule.

Human tRNAGlu genes: their copy number and organisation

The tRNAGlu gene copy number, determined by genomic blot analysis of human plaeental 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 λ Charon‐4A recombinants of human genomic DNA containing two different tRNAglu genes.

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.

Non-enzymic binding of phenylalanyl transfer ribonucleic acid to ribosomes and ribosomal subunits from rat liver

1. 1. (3H)phe-(32P)tRNA was used to compare the binding of phe-tRNA to 80 S ribosomes and 40 S subunits from rat liver in circumstances where deacylation of bound tRNA could be measured. 2. 2. About twice as much phe-tRNA was bound to the 80 S ribosome as to the 40 S subunit. 3. 3. Deacylation of phe-tRNA bound to the 80 S ribosome did not occur but about 20 % of the phetRNA bound to the 40 S subunit was lost by deacylation.