Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).     

The analysis of hypervariable DNA profiles: problems associated with the objective determination of the probability of a match

Summary A computerised system has been used to store DNA profiles from 3 hypervariable loci. This initial survey illustrates that band matching is only possible after analysis of the errors associated with electrophoretic systems. A number of databases have been constructed with the three probes investigated and two methods of frequency determination, binning and sliding window fitting, have been compared.     

AN AMPLIFICATION OF THE PHYLOGENETIC SPECIES CONCEPT

Abstract— The goal of a phylogenetic species concept is to reveal the smallest units that are analysable by cladistic methods and interpretable as the result of phylogenctic history. We define species as the smallest aggregation of populations (sexual) or lineagcs (asexual) diagnosable by a unique combination of character states in comparable individuals (semaphoronts). A character state is an inherited attribute distributed among all comparable individuals (semaphoronts) of the same historical population, clade, or terminal lineage. This definition of species is character-based and pattern oriented. Evolutionary explanations of phylogenetic species are consistent with contemporary explanations of processes of speciation, but require only the assumption of nested hierarchical pattern. We discuss the compatibility of the phylogenetic species concept with various biological needs for species and justify its use at the exclusion of alternative species concepts.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

The importance of Paracallisoma coecus and myctophid fishes to nesting fork-tailed and Leach's storm-petrels in the Queen Charlotte Islands, British Columbia

The diet of fork-tailed (Oceanodroma furcata) and Leach's storm-petrel(O.leucorhoa) was studied at Petrel Island, a small island northof Hippa Island in the Queen Charlotte Islands, British Columbiain 1983. The diet of the two storm-petrel species consistedmostly of the amphipod Paracallisoma coecus and fish. Most fisheswhich could be identified were myctophids, and may have beenacquired by storm-petrels at night. Fishes increased while P.coecusdeclined in importance as a food over the nestling season ofthe storm-petrels. The amount of oil also declined in the Leach'sstorm-petrel diet over the season. Since P.coecus contains muchoil, the seasonal decline of both P.coecus and oil was probablyassociated. Other less important foods were jellyfishes, copepods,isopods, euphausiids, shrimp, squid and octopus. Jellyfishes(Velella velella) only occurred in the diet of Leach's storm-petrels,which may relate to that storm-petrel feeding in open oceanwhere V. velella occurs most frequently. The most numerous copepodwas Neocalanus cristatus, which was only observed in the storm-petreldiet early on during the study period. An abyssal copepod, Bathycalanusbradyi, and the isopod Cirolana californiensis (tentativelyidentified, the latter only previously known from sea canyonsoff California) were surprising food items of storm-petrels.Most euphausiid prey consisted of Thysanoessa spinifera andEuphausia pacifica.     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.