On the statistical assessment of similarities in DNA sequences

The statistical behavior of the similarity score for unrelated DNA sequences calculated as letter-by-letter comparison or from various forms of optimal alignment was studied. It was found that natural DNA-sequences from a data base and true random sequences show the same statistical behavior in terms of such scores. This makes it possible to adopt a simple criterion for the rejection of fortuitous similarity. It is based on the mean and standard deviation of chance scores whose expected values, depending on chain length, gap penalty and probability of letter coincidence, may be calculated from formulae given in the paper.     

Physical map of the bacteriophage L (Salmonella typhimurium)

Abstract A circular restriction map of the genome of the phage L ( Salmonella typhimurium ) has been constructed with five restriction endonucleases, Eca I, Eco RI, Bam HI, Bgl I, and Pst I. The Eco RI fragments of phage-L DNA were cloned into pACYC184, and the resulting recombinant plasmids pL1, pL2,…,pL7 were introduced into Salmonella typhimurium . The genes present on the fragments cloned were identified by the marker rescue experiments with the L-phage amber mutants. A physical gene map of the L genome obtained in this way was compared with that of P22.     

Inbreeding and the genetic control of nondisjunction

Summary We have studied the frequency of trisomics in newly formed zygotes and the proportion of trisomics, k, coming from consanguineous marriages by assuming that recessive genes at a single locus or multiple loci are responsible for the induction of nondisjunction. For mitotic nondisjunction, the value of k increases as the magnitude of consanguinity of the parents increases, but the opposite relationship holds for meiotic nondisjunction. Therefore, it is important to distinguish mitotic and meiotic types in the genetic study of nondisjunction. This seems to be one of the simples tests for detecting the genetic control of nondisjunction.     

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma

Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation. This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National Bladder Cancer Project and by the Medical Research Service of the Veterans Administration.     

Indication of the metarhodopsin I–II transition by absorption-changes of eriochromblack T

The dye eriochromblack T (erio T), added to an aqueous suspension of bovine retinal outer segments solubilized by digitonin, shows a light-induced absorption-increase at =645 nm. Erio T is shown to directly interact with micellar metarhodopsm I and metarhodopsin II. The absorption-changes of erio T can be regarded as an indication of the transition from the metarhodopsin I conformation (with associated Ca²⁺) to the metarhodopsin II conformation (with associated H⁺).Thanks are due to the Deutsche Forschungsgemeinschaft for financial support (Em 18/1-4).     

In vitro specificity of EcoRI DNA methyltransferase.

The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap.     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.