Expression and analysis of the gene for the catalytic beta subunit of the sodium-translocating NADH-ubiquinone oxidoreductase of Vibrio alginolyticus.
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Amino Acid Sequence↗
Biomedical subjects
D R Leach
Publications and source records attributed to D R Leach.
The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination.
BACKGROUND: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome-mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants. RESULTS: We have purified the E. coli SbcCD protein from an overexpressing strain and have shown that it has an ATP-dependent DNA double-strand exonuclease activity. Co-purification of nuclease with protein, antibody inhibition and the absence of activity in extracts lacking the sbcCD genes confirm that the activity is intrinsic to SbcCD. The purified protein also has an ATP-independent single-strand DNA endonuclease activity. CONCLUSIONS: We have shown that sbcCD encodes a nuclease. The purified protein has double-strand DNA exonuclease and single-strand DNA endonuclease activities. We propose that SbcCD cleaves secondary structures formed at replication forks and that the broken forks can be repaired by homologous recombination.
Adenosine Triphosphate↗
Palindrome-induced deletion in enterobacterial repetitive sequences.
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Base Sequence↗
Tumor cells engineered to express major histocompatibility complex class II molecules induce T helper cell-dependent responses that protect mice from normally lethal doses of unmodified tumor cells.
Tumor cells typically fail to stimulate protective immune response in the autochthonous host. This does not appear to be the result of either inadequate antigenicity or failure to express a normal complement of major histocompatibility complex (MHC) class 1 molecules. To investigate if tumor cells fail to stimulate protective immunity because they fail to activate adequate numbers of T helper cells, we transfected murine fibrosarcoma and melanoma cells with genes encoding syngeneic and allogeneic MHC class II molecules. Fibrosarcoma cells expressing either type of MHC class II molecules failed to induce tumors in syngeneic mice and stimulated T helper cell-dependent antitumor immune responses that protected mice from subsequent challenge with untransfected tumor cells. The antitumor response involved both CD4+ and CD8+ T cells, and appeared to be dependent on at least low levels of innate tumor cell immunogenicity.
Animals↗
DNA palindromes adopt a methylation-resistant conformation that is consistent with DNA cruciform or hairpin formation in vivo.
Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli. It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences. This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive. If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of a long perfect palindrome located in bacteriophage lambda is shown to be methylation-resistant in vivo. Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre. We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context. These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome.
Bacteriophage lambda↗
Fibrosarcoma cells expressing allogeneic MHC Class II antigens induce protective antitumor immunity.
The initiation of effective immune responses usually requires presentation of Ags by MHC class I and class II molecules. Although most tumors express MHC class I molecules, MHC class II molecule expression is generally limited to specialized APCs. One reason spontaneous tumors may fail to elicit effective immune responses is that tumor Ags are inefficiently presented by APCs, and adequate T cell-mediated help is not generated. To test this hypothesis, we investigated the possibility of enhancing Th cell stimulation by inducing expression of MHC class II molecules on tumor cells. We transfected a murine fibrosarcoma, Sa1N, with the genes encoding allogeneic (I-Ad) or syngeneic (I-Ak) MHC class II molecules. We then compared the tumorigenic and immunogeneic potential of these transfectants to parental Sa1N tumor cells. Subcutaneous injection of allogeneic or syngeneic transfectants resulted in dramatically fewer tumors than injection of unmodified fibrosarcoma cells, and mice inoculated with MHC class II gene-transfected cells were resistant to subsequent challenge with parental Sa1N cells. Rejection of allogeneic MHC class II Ag+ tumor cells could be mediated by either CD4+ or CD8+ T cells, whereas rejection of secondary challenge with wild-type Sa1N tumor cells required both T cell subsets. These results demonstrate that allogeneic, as well as syngeneic, MHC class II Ag+ tumor cells can stimulate protective antitumor immunity.
Animals↗
Manipulation of costimulatory signals to enhance antitumor T-cell responses.
One of the major goals of tumor immunotherapy is the induction of tumor-specific T-cell responses that will be effective in eradicating disseminated tumors. Emerging information on the role of costimulatory molecules in T-cell activation offers several new strategies for enhancing antitumor responses, including the induction of expression of costimulatory molecules on tumor cells, enhancement of the presentation of transferred tumor antigen by host antigen-presenting cells, and ex vivo antigen priming of autologous antigen-presenting cells.
Animals↗
The effects of trinucleotide repeats found in human inherited disorders on palindrome inviability in Escherichia coli suggest hairpin folding preferences in vivo.
Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that are associated with several human inherited disorders. We present evidence consistent with the folding of these trinucleotide repeats into hairpin loops at the center of a long DNA palindrome in vivo. Our assay utilizes a palindrome in bacteriophage lambda, the center of which determines its ability to inhibit plaque formation in a manner that is consistent with folding into a hairpin or cruciform structure. We show that central inserts of even numbers of d(CAG).d(CTG) repeats inhibit plaque formation more than do odd numbers. Both d(CAG)2.d(CTG)2 and d(CGG)2.d(CCG)2 central sequences behave like DNA sequences known to form two-base loops in vitro, suggesting that they may also form compact and stable loops. By contrast, repeats of d(GAC).d(GTC) do not show any evidence consistent with unusual loop stability. These results agree with in vitro evidence that the unstable repeats can form hairpin secondary structures and suggest a favored position of folding. We discuss the potential roles of secondary structures, DNA replication and recombination in models of repeat tract expansion.
Bacteriolysis↗
Structural and functional similarities between the SbcCD proteins of Escherichia coli and the RAD50 and MRE11 (RAD32) recombination and repair proteins of yeast.
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Amino Acid Sequence↗
Protective antitumor immunity induced by immunization with MHC class II gene-transfected tumor cells is unrelated to MHC class II expression.
A/JCr mice reject Sa1N fibrosarcoma cells genetically engineered to express major histocompatibility complex (MHC) class II molecules and are highly resistant to subsequent challenge with unmodified Sa1N cells. In this report we examine the mechanism by which this protective antitumor immunity is induced. We found that MHC class II antigen-positive tumor cells were no more effective than irradiated, MHC class II antigen-negative cells at inducing secondary protective immunity. Additionally, therapeutic immunization with MHC class II antigen-positive tumor cells had no effect on the growth of admixed Sa1N cells or preexisting Sa1N tumors. Based on these observations, we conclude that the MHC class II antigen-induced immune response is not related to Sa1N-specific antitumor immunity.
Animals↗
Two-base DNA hairpin-loop structures in vivo.
In vitro studies have revealed that DNA hairpin-loops usually contain four unpaired bases. However, a small subset of sequences can form two-base loops. We have previously described an in vivo assay that is sensitive to tight loop formation and have set out to test whether DNA sequences known to form two-base loops in vitro also form tight loops in vivo. It is shown that the sequences 5'dCNNG and 5'dTNNA behave as predicted if they favour two-base loop formation in vivo, a result that is consistent with previously described in vitro studies. The ability of specific DNA sequences to form tight loops in vivo has implications for their potential to form transient structures involved in gene regulation, recombination and mutagenesis.
Bacteriophage lambda↗
Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair.
Long DNA palindromes pose a threat to genome stability. This instability is primarily mediated by slippage on the lagging strand of the replication fork between short directly repeated sequences close to the ends of the palindrome. The role of the palindrome is likely to be the juxtaposition of the directly repeated sequences by intra-strand base-pairing. This intra-strand base-pairing, if present on both strands, results in a cruciform structure. In bacteria, cruciform structures have proved difficult to detect in vivo, suggesting that if they form, they are either not replicated or are destroyed. SbcCD, a recently discovered exonuclease of Escherichia coli, is responsible for preventing the replication of long palindromes. These observations lead to the proposal that cells may have evolved a post-replicative mechanism for the elimination and/or repair of large DNA secondary structures.
Base Sequence↗
The effects of nucleotide sequence changes on DNA secondary structure formation in Escherichia coli are consistent with cruciform extrusion in vivo.
The construction in bacteriophage lambda of a set of long DNA palindromes with paired changes in the central sequence is described. Identical palindrome centers were previously used by others to test the S-type model for cruciform extrusion in vitro. Long DNA palindromes prevent the propagation of carrier phage lambda on a wild-type host, and the sbcC mutation is sufficient to almost fully alleviate this inviability. The plaque areas produced by the palindrome containing phages were compared on an Escherichia coli sbcC lawn. Central sequence changes had a greater effect upon the plaque area than peripheral changes, implying that the residual palindrome-mediated inviability in E. coli sbcC is center-dependent and could be due to the formation of a cruciform structure. The results argue strongly that intrastrand pairing within palindromes is critical in determining their effects in vivo. In addition, the same data suggests that DNA loops in vivo may sometimes contain two bases only.
Bacteriophage lambda↗
The effects of central asymmetry on the propagation of palindromic DNA in bacteriophage lambda are consistent with cruciform extrusion in vivo.
The propagation of lambda phages carrying long perfect palindromes has been compared with that of phages carrying imperfect palindromes with small regions of central asymmetry. The perfect palindromes confer a more deleterious phenotype than those with central asymmetry and the severity of the phenotype declines with the length of asymmetry in the range from O to 27 base pairs. These results argue that a center-dependent reaction is involved in the phenotypic effects of palindromic DNA sequences, consistent with the idea that cruciform extrusion occurs in vivo.
Bacteriophage lambda↗
The SbcCD protein of Escherichia coli is related to two putative nucleases in the UvrA superfamily of nucleotide-binding proteins.
The derived amino-acid sequences of the proteins encoded by E. coli genes sbcC and sbcD have been compared with other protein sequences using computer assisted methods. This work has shown that SbcC and D, which inhibit the propagation of replicons containing long palindromic DNA sequences, are distantly related to two putative bacteriophage nucleases. These nucleases both comprise two polypeptide chains which are the products of genes 46 and 47 of bacteriophage T4 (gp 46 and gp 47) and genes D13 and D12 of bacteriophage T5 (gp D13 and gp D12). The comparisons reveal that SbcC, gp 46 and gp D13 are more closely related to each other than are SbcD, gp 47 and gp D12. SbcC appears to have undergone a partial duplication of an ancestral sequence. These proteins all contain motifs common to the superfamily of nucleotide-binding proteins that includes UvrA and the cystic fibrosis transmembrane regulator CFTR.
Adenosine Triphosphatases↗
Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12.
The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate lambda red gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic lambda phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.
Bacteriophage lambda↗
Persistent cryptosporidiosis in horses with severe combined immunodeficiency.
Cryptosporidial infections were established in five young foals with severe combined immunodeficiency following oral administration of 10(8) Cryptosporidium parvum oocysts. All foals shed oocysts (average of 8 x 10(6) to 2 x 10(8)/g of feces) until death. Inflammation and C. parvum organisms were observed in the common bile duct, duodenum, jejunum, and ileum. Since foals with severe combined immunodeficiency lack functional T and B lymphocytes and are incapable of antigen-specific immune responses, they are well suited for evaluating the pathogenesis and treatment of persistent cryptosporidiosis.
Animals↗
Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition.
We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.
Bacteriophage mu↗