Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes.

The soybean possesses a gene family encoding the major low mol. wt. heat‐shock proteins of 15–18 kd. We have determined the primary DNA sequences of two of the genes, both located on the same subgenomic DNA fragment. The protein coding regions are characterized by long uninterrupted open reading frames and by sequence homology of 92% and 100% with a heat‐shock specific cDNA. One protein sequence deduced from the completely cloned gene hs6871 is composed of 153 amino acids with a total mol. wt. of 17.3 kd; the other protein is a truncated polypeptide containing 73 amino acids at the carboxy‐terminal end of an incompletely cloned heat‐shock gene designated hs6834. Investigations of the hydrophilic/hydrophobic characteristics of the polypeptides revealed a conservation of structural features between heat‐shock proteins from soybean, Caenorhabditis and Drosophila and mammalian lens α‐crystallin. The 5′ end of the soybean heat‐shock gene hs6871 was mapped by S1 nuclease at a position which is ˜100 nucleotides upstream from the translation start codon and 25 nucleotides downstream from a TATA‐box sequence. Six other potential promoter elements which are homologous to the Drosophila heat‐shock consensus sequence CT‐GAA‐TTC‐AG‐, are present within ˜150 nucleotides upstream from the TATA‐box. The possible functions of these promoter elements in transcriptional regulation of expression of soybean heat‐shock gene are discussed.

The 5' DNA sequences involved in the thermal inducibility of the soybean heat shock gene hs6871 were analysed in transgenic tobacco plants. The transcriptional activity of various in vito generated deletion mutants was examined by Northern blot analysis, Si nuclease mapping and dot-blot hybridization. At least 181 bp upstream from the translational start site are sufficient for thermal induction at 40°C and correct initiation of transcripts. Full promoter activity with the induction of wild-type levels of transcripts requires additional upstream sequences contained within 439 bp 5' to the coding sequence. Our results suggest that faithful regulation and the generation of high levels of hs6871-specific mRNA depend on the presence of sequences which show homology to the 14-bp heat shock consensus element of Drosophila and, in addition, on as yet unidentified enhancer-like upstream sequences.

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We present in Table I an updated list of the sensitivities of 298 restriction endonucleases and 20 DNA methyltransferases to sitespecific modification at 4-methylcytosine (m4C), 5-methylcytosine ('OC), 5-hydroxymethylcytosine (hm5C), and 6-methyladenine (m6A) (McC 14), four modifications that are common m the DNA of prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcl 1,Ne3,Ne4). In addition, new information is included on restriction endonuclease cleavage at sites modified with 5-hydroxymethyluracil (h'5U). Knowledge of the sensitivity of restriction endonucleases to site-specific modification can be used to study cellular DNA methylation. Several restriction-modification enzymes share the same recognition sequence specificity, but have different sensitivities to site-specific methylation. Table II lists 32 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ. The data presented here and an additional three other tables are available in printed form or as a text file on a 3.5" Macintosh diskette. The extra tables include Table III which is a list of over 205 characterized DNA methyltransferases. A detailed list of cloned restriction-modification genes has been made by Wilson (Wi4). Table IV lists the sensitivities of over 20 Type II DNA methyltransferases to m4C, m5C, hm5C, and m6A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu9,Mc10, Ne3,Po4), and this sensitivity often differs from that of their restriction endonuclease partners. Table V gives a list of restriction systems in this review alphabetized by recognition sequence. The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CIBR, phone 619 535 5486, FAX 619 535 5472. Molecular basis for sensitivity restriction enzymes to methylation m4C, m5C, hm5C, and m6A are bulky alkyl substitutions in the major groove of DNA. Site-specific DNA methylation can interfere with many sequence-specific DNA binding proteins (e.g. St2,Wa8), including binding of restriction endonucleases and DNA methyltransferases. At the molecular level, the inability of restriction enzymes to cut modified DNA can be explained using EcoRI and EcoRV endonucleases as instructive models. DNA modification can interfere with substrate binding and/or conformational changes of the enzyme: substrate complex. Based on the EcoRI: DNA co-crystal structure (MclS,Ro8), methylation of either adenine (Gm6AATTC or GAm6ATTC)