The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning

A mouse cDNA library derived from the EL4 cell line (b-haplotype) was screened with a probe containing a small part of the H-2K^b coding region. One of the clones isolated, pH203, encodes a protein whose deduced amino acid sequence is identical with the known sequence of H-2D^b in 141 of 141 positions available for comparison. The clone, therefore, is believed to code for the H-2D^b transplantation antigen. The cDNA insert of pH203 contains the coding region for residues 82 through the carboxy-terminus of H-2D^b, and includes 476 nucleotides of the 3-untranslated sequence. Comparison between the H-2D^b cDNA clone and a previously isolated H-2K^b cDNA clone shows homologies of 83% and 91% at the amino acid and nucleotide levels, respectively. Analysis of DNA sequences at the 3-coding and untranslated regions suggests that the mRNAs of H-2K^b and H-2D^b are spliced differently at their 3-coding ends.     

Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning

Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70°C.Issued as National Research Council of Canada No. 30545     

Identification,molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens

The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.     

Monoclonal anti-idiotypic antibodies to an I-J interacting molecule inhibit suppression in an H-2 restricted way

We have obtained 10 mAb from two independent fusions that are anti-idiotypic to the combining site of an anti-I-Jk antibody. These mAb block Ts cell function isn a genetically restricted manner in vitro and in vivo and recognize a determinant on macrophage membranes. In addition, they do not affect the I-Ek-restricted activation of a Th cell line specific for pigeon heart cytochrome c. We conclude that these mAb may recognize a molecule other than conventional I-Ek on cells interacting with Ts cells that is involved in mediating Ts activity. The molecule recognized may be a modified I-Ek molecule or a molecule not encoded by the genes encoding I-Ek.     

Identification and molecular cloning of functional chicken IL-12

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.     

Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning

Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.     

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.     

Detection of H-2K mRNA in mouse 8-cell embryo by cDNA cloning

Mouse MHC class I gene expression in 8-cell embryo was examined by cDNA cloning. We constructed a cDNA library from 8-cell embryos of ICR mice and isolated a class I cDNA from 3.0 x 10(5) phage clones of the library. Sequencing analysis of this clone revealed it to include the cDNA fragment extending from the exon 6 of the cytoplasmic portion to 3' untranslated region 1 of H-2K gene. Qa, Tla or other embryonic class I cDNA have not been isolated in the library.     

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3'' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.     

Extension of the H-2 TLb molecular map

A region of the TL ^b locus encompassing T11 to T13 contains retroviral sequences TLev1 and TLev2. As part of a study to determine whether the retroviral elements are involved in the expression of TL genes, the genomic organization of this region was reexamined in greater detail. A result of these investigations is the extension of the H-2 TL^b molecular map. Two additional TL genes have been isolated from C57BL/6 mice, T14 and T15. The genomic organization of T9 through T15 is presented. The nucleotide sequence has been determined for exons 4, 5, and 6 of T13. As a result of a C to T conversion, a termination codon is introduced into exon 4, indicating that T13 either encodes a secreted protein or is a pseudogene. T13 was found to be more homologous to the H-2 genes outside the TL region. T14 has been physically disrupted by the integration of TLevl , and the H-2 sequences appear to have diverged greatly. The relationship of the TL regions of the b and c haplotypes has been investigated using numerous low copy probes. The genome of BALB/c (TL^c) is shown to lack a counterpart of the T13–T15 ^b region. Homologous regions exist in the two haplotypes; yet considerable polymorphism is observed. TL^b mice do not express TLa on the cell surface of normal thymocytes while TL^c mice do; TLa expression is activated in many TO leukemias. The diversity seen in the T13–T15 region may provide insights into the phenotypic expression or regulatory mechanisms of TL expression in these two haplotypes.     

A novel receptor on allograft (H-2d)-induced macrophage (H-2b) toward an allogeneic major histocompatibility complex class I molecule, H-2Dd, in mice

The generation of knockout mice demonstrated that noncytotoxic CD4(+), but not cytotoxic CD8(+), T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograftinduced macrophages (AIM) in mice lysed allografts with H-2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H-2D(b)) for an allogeneic major histocompatibility complex (MHC) class I molecule, H-2D(d), by using H-2D(d) tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181-bp) encoded a 342-amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H-2D(d), but not H-2L(d), H-2K(d), H-2D(b), H-2K(b), H-2D(k), or H-2K(k), molecules; and the H-2D(d) binding was suppressed by the addition of R15 or anti-H-2D(d) mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H-2D(d), but not H-2D(b) or H-2D(k), molecules. The dissociation constant of AIM toward H-2D(d) tetramers was 1.9 x 10(-9) M ; and the binding was completely inhibited by the addition of R15 or anti-H-2D(d) mAb. These results reveal that a novel receptor for an allogeneic H-2D(d) molecule was induced on effector macrophages responsible for allograft (H-2(d)) rejection in H-2(b) mice.     

NMR studies of an oligoproline-containing peptide analogue that binds specifically to the H-2Kd histocompatibility molecule

T lymphocytes expressing variable cell surface antigen receptors recognize "processed" forms of antigen, presented on the surface of other cells by molecules of the major histocompatibility complex (MHC). Naturally processed antigenic peptides can be replaced by synthetic ones. The synthetic peptide AYPPPPPTLA (P5) is an active competitor to the antigenic peptide HLA A24 170-182 (sequence RYLENGKETLQRA) that is recognized by A24 specific T cells in association with the H-2Kd class I MHC molecule. In P5 the five prolines were designed to play the role of a rigid spacer between the residue Y and the T-L unit, so as to mimic the role of Y171, T178, and L179 in the HLA A24 antigenic peptide, since these residues have proven to be the most important with respect to the binding of the HLA A24 peptide with the H-2Kd MHC molecule. Nuclear magnetic resonance studies allow us to demonstrate that in aqueous solution P5 adopts at least three long-lived conformations that can be classified with respect to the Y2-P3-P4 amide bonds as trans-trans, cis-trans, and cis-cis. Among these, the trans-trans form is present in 67% of the molecules while the two others share the remaining 33%.     

An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.     

Biochemical and functional analyses of a secreted H-2Ld molecule.

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.     

Identification of nonplanar small molecule for G-quadruplex grooves: molecular docking and molecular dynamic study

DNA G-quadruplex is an attractive drug target for anticancer therapy. Most G-quadruplex ligands have little selectivity, due to π-stacking interaction with common G-tetrads surface. Thanks to the varieties of G-quadruplex grooves, the groove-binding ligand is expected to create high selectivity. Therefore, developing novel molecular geometries that target G-quadruplex groove has been paid growing attention. In this work, steroid FG, a special nonplanar and nonaromatic small molecule, interacting with different conformations of G-quadruplexes has been studied by molecular docking and molecular dynamics simulations. The results showed the selectivity of the hydrophobic group of steroid FG for the wide groove of antiparallel G-quadruplex. The methyl groups on the tetracyclic ring of steroid represent the specific binding ability for the small hydrophobic cavity formed by reversed stacking of G-tetrads in antiparallel G-quadruplex groove. This work provides new insight for developing new classes of G-quadruplex groove-binding ligands.