A computer algorithm to determine the recognition site of restriction enzymes

A new algorithm is proposed to determine the type-II restrictionendonucleases' recognition site knowing the digested DNA sequenceand fragment lengths in an actual case. The algorithm is implementedfor the Commodore 64 microcomputer. Received on January 6, 1987; accepted on June 19, 1987     

A study of free radicals in paraffin embedded and deparaffinized human heart muscle tissue using electron spin resonance (ESR)

Summary Free radicals (spectroscopic splitting factor; g factor=2.003–2.005) were investigated in formol-fixed, paraffin embedded heart-muscle tissue sections using electron spin resonance (ESR) spectra. Changes in signal amplitude, g factor and line width were registered during deparaffinization, chloroform-methanol extraction, vapour treatment and bromination. An attempt was made to identify the source of the ESR signals by a correlation between the signal amplitude and number of fluorescent and/or Sudan-black-positive granules counted in the tissue sections. An increase in signal amplitude, g value and line narrowing were characteristic of the ascorbyl radical after deparafinization in air. Vapour treatment revelated that the broader signal has lower g factor, a characteristic that is tentatively assigned to oxidized lipids. The bromination resistant minor fraction of free radical centres with small g factor might be associated with the pigment content of the samples.     

Identification and Characterization of a cDNA and the Structural Gene Encoding the Mouse Epithelial Membrane Protein-1

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.     

CD and Fourier transform ir spectroscopic studies of peptides. II. Detection of β-turns in linear peptides

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) β-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of β-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the β-turn band shewed up between 1639 and 1633 cm^?1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band war-also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3₁₀-helical polypeptides and protein in D₂O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508–512; H. H. Mantsch, A. Perczel. M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch. A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers. Vol. 33, pp. 201–207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Tedd, and G. L. Millhauser (1992). Nature, Vol. 359, pp. 653–655], suggest that the amide I band, with a major contribution from the acceptor C ? O of the 1 ← 4 intramolecular H bond of β-turns, appears near or below 1640 cm^?1, rather than above 1660 cm^?1. In TFE, bands between 1670 and 1660 cm^?1 are mainly due to “free” carbonyls, that is, C ? O's of amides that are solvated but not involved in the characteristic H bonds of periodic secondary structures or β-turns. © 1994 John Wiley & Sons, Inc.     

Evaluation of “sequence-tagged-site” PCR products as molecular markers in wheat

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.Contribution no. J-2833 of the Montana Agric Exp Stn     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.