Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new type of illegitimate recombination is dependent on restriction and homologous interaction.

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.     

Human T cell recognition of influenza A nucleoprotein. Specificity and genetic restriction of immunodominant T helper cell epitopes.

The characterization of human T cell antigenic sites on influenza A nucleoprotein (NP) is important for subunit vaccine development for either antibody boosting during infection or to stimulate T cell-mediated immunity. To identify such sites, 31 synthetic peptides that cover more than 95% of the amino acid sequence from NP of influenza A/NT/60/68 virus were tested for their ability to stimulate PBL from 42 adult donors. The most frequently recognized region was covered by a peptide corresponding to residues 206-229 of NP, with 20/42 (48%) of responders. In many individuals this was also one of the peptides that stimulated the strongest T cell responses. Other regions that were also frequently recognized were 341-362 by 13/42 (30%), 297-318 by 10/42 (23%), and 182-205 by 9/42 (21%) of individuals. These peptides covered highly conserved regions in NP of influenza A viruses, suggesting that they could be useful in boosting cross-reactive immunity against the known type A virus strains. In order to define the class II restriction molecules used to present particular T cell epitopes, 22 persons from the donor panel were HLA-typed. The majority of persons who expressed DR2, and proliferated to NP also responded to the major immunodominant region 206-229. In addition, this peptide was also immunodominant in the one person expressing DRw13. The observation that recognition of this peptide is associated with DR2 was confirmed by using short term T cell lines and APC from a panel of typed donors. Further results with virus-specific T cell lines and clones and transfected L cells expressing DR molecules showed that DR1 could also present this peptide. Therefore the results suggest that recognition of 206-229 is associated with at least three different DR haplotypes and this may explain the high frequency with which this peptide is recognized in the population. The fine specificity of the response to peptide 206-229 was distinct when presented by DR1- or DR2-expressing APC. The DR1 response was dependent on the N terminus, and the DR2 response was directed to the C terminus of the peptide.     

MHC interaction and T cell recognition of carbohydrates and glycopeptides.

The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells.     

Recombination in bacteriophage T1 in the presence of host restriction.

When unmodified phage T1 infects restricting host cells at high multiplicities of infection, there is an increase in recombination frequency in all regions of the T1 map compared to the level of recombination in standard crosses when short distances are examined. The enhancement of recombination frequency is not uniform for all regions but is greatest for markers near the center of the map and not so great for markers near the ends. Crosses between markers at the extremities of the map show that there is no increase in recombination frequency under restriction conditions. An examination of phage T1 heterozygotes suggests that an increase of ends created by the process of P1 restriction increases recombination. When T1 crosses are done in the absence of host restriction, recombination defects in the host have no effect on phage recombination and we conclude that phage T1 codes for its own recombination genes. Host recombination functions are also dispensable for the recombination occurring during infection of restricting host cells by unmodified phage at high multiplicities of infection.     

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.     

Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.     

Antigen-specific clones of proliferating T lymphocytes. I. Methodology, specificity, and MHC restriction

In this report we describe in detail a new method for cloning antigen-specific, proliferating T lymphocytes directly from primed murine lymph nodes after 3 days of activation in vitro. After expansion in liquid culture the cells from the colonies were shown to be antigen specific and to require I-A histocompatible, irradiated spleen cells for stimulation. For hapten-carrier-type antigens, the T cells were shown to be carrier specific in their recognition but they were also capable of distinguishing the presence of the hapten. Recloning of small numbers of these cells in soft agar under conditions of high plating efficiency yielded true clones (i.e., populations derived from a single cell) whose antigen specificity was identical to that of cells from the original colony. The fact that a clone of T cells was I-A restricted in its antigen recognition demonstrates that suppressor T cell function cannot account for the phenomenon of major histocompatibility complex restriction.     

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA.

A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, KpnI, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.     

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.

Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.     

Different restriction of bacteriophages T3 and T7 by P1-lysogenic cells and the role of the T3-coded SAMase.

The intracellular growth of the phages T3 and T7 is restricted in the presence of the Escherichia coli prophage P1. Phage T3 has a higher ability to express its genome and to damage the host cell than T7. This partial protection of T3 against P1 restriction is due to the T3-coded SAMase, an enzyme which degrades S-adenosylmethionine, the cofactor of the P1 restriction endonuclease. Since we did not observe DNA cleavage in vivo, we conclude that the in vivo action of the P1 nuclease is limited to a SAM-dependent repressor-like binding to T3 and T7 DNA, while further reactions with the DNA (modification vs cleavage) are blocked.     

Physical mapping of the restriction fragments obtained from bacteriophage T4 dC-DNA with the restriction endonucleases SmaI, KpnI and BglII.

Summary The cytosine-containing DNA of a mutant of bacteriophage T4 was digested with restriction endonucleases SmaI, KpnI and BglII producing 5, 7 and 13 fragments respectively. Complete physical maps of the T4 genome were constructed with the enzymes SmaI and KpnI and an almost complete map with the enzyme BglII.     

HLA class II restriction of T helper cell response to cytomegalovirus (CMV). I. Immunogenetic control of restriction

All three HLA class II families (DR, DQ, and DP) are involved in restriction of helper T cell (Th) recognition of nominal antigens including CMV. Only limited studies have been described previously to determine whether restricting determinants of DR and especially DQ are subtypic to the serologically defined DR and DQ specificities, and to what extent restricting determinants are associated with Dw specificities defined in alloresponses. In the present report, we describe a large number of CMV-specific Th clones derived from two different individuals who are seropositive for CMV. Clones were classified as being DR-, DQ-, or DP-reactive based on blocking with monoclonal antibodies. DR- and DQ-restricted clones were then examined in panel studies using antigen-presenting cells (APC) expressing the Dw subtype of the restricting DR-DQ haplotype, as well as APC expressing different Dw subtypes associated with the serologically defined specificity. Unrelated specificities were also included. Our findings show that not only for DR but for DQ as well, the primary restricting determinants appear to be subtypic to the serologically defined antigen; furthermore, subtype restriction for both DR and DQ is very closely associated with single Dw specificities. In several cases in which cross-reactivity among restricting Dw specificities was observed in association with a given DR or DQ haplotype, a molecular basis could be suggested to explain the cross-reacting determinants. A small minority of the clones appeared to be CMV specific, but was restricted by a determinant(s) that is either monomorphic or minimally polymorphic.     

Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1.

Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C. Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA. Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA. Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis. Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E. coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present at 42 degrees C.     

Development of Escherichia coli virus T1: escape from host restriction.

The bacterial virus T1 grows interchangeably on different Escherichia coli strains (C, B, and K). This implies that T1 has an efficient mechanism to overcome the host restriction barrier. The DNA of T1 was found to be methylated independently of the hosts. The percentage of N6-methyladenine varied from 1.6 to 1.8, and the 5-methylcytosine content varied from 0.1 to 0.4%. In contrast, the range in percentage of N6-methyladenine and 5-methylcytosine found in the hosts was 0.7 to 2.4 and 0.0 to 1.1, respectively.     

SAMase gene of bacteriophage T3 is responsible for overcoming host restriction.

Deletion and point mutants of T3 have been isolated and used to show that the early region of T3 DNA is organized in the same way as that of T7 DNA. Homologous early RNAs and proteins of the two phages have been identified by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Both phages have five early mRNA's, numbered 0.3, 0.7, 1,1.1 and 1.3 from left to right, although no T3 protein that corresponds to the 1.1 protein of T7 has yet been identified. In general, corresponding early RNAs and proteins of the two phages migrate differently on gels, indicating that they differ in molecular weight and/or conformation. In both T7 and T3, gene 0.3 is responsible for overcoming the DNA restriction system of the host, gene 0.7 specifies a protein kinase, gene 1 specifies a phage-specific RNA polymerase, and gene 1.3 specifies a polynucleotide ligase. The 0.3 protein of T3 is responsible for the S-adenosylmethionine cleaving activity (SAMase) induced after T3 (but not T7) infection. However, cleaving of S-adenosylmethionine does not appear to be the primary mechanism by which T3 overcomes host restriction, since at least one mutant of T3 has lost the SAMase activity without losing the ability to overcome host restriction.