bluuebunny/crossref_metadata_2025 huggingface dataset:
I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.
Authors: Bell-Pedersen D , Quirk S M , Bryk M , Belfort M
Published: May 2006

Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.
Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.
Authors: Li C , Lai C F , Sigman D S , Gaynor R B
Published: May 2006

Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements.
Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.
Authors: Wickner S , Hoskins J , McKenney K
Published: May 2006

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp70 in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication. Activation is the conversion of RepA dimers into monomers in an ATP-dependent reaction and the monomer form binds with high affinity to oriP1 DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate oriP1 DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. Based on our data and previous work, we present a model for heat shock protein action under normal and stress conditions.
Somatic mutation in constant regions of mouse lambda 1 light chains.
Authors: Motoyama N , Okada H , Azuma T
Published: May 2006

To study the distribution of somatic mutation, we determined nucleotide sequences of rearranged lambda 1-chain genomic DNA from four hybridomas obtained from C57BL/6 mice that had been immunized with (4-hydroxy-3-nitrophenyl)acetyl-conjugated chicken gamma globulin. In total, 114 nucleotide substitutions were observed, with neither insertion nor deletion. Sixty-one mutations occurred in the variable-joining region genes (V lambda 1-J lambda 1) and 49 in joining-constant (J lambda 1-C lambda 1) introns. Although frequency decreased with distance from the V lambda 1-J lambda 1 coding region, somatic mutations occurred in the entire J lambda 1-C lambda 1 intron and even in the C lambda 1 region. We found four nucleotide substitutions in C lambda 1 genes, all of which were replacement mutations. Therefore, the mechanism responsible for somatic mutation is operative into the C lambda 1 exons. Nucleotide sequences of rearranged but inactive lambda 2-chain genes from two hybridomas were also examined and compared with those of lambda 1-chain genes. The clustering of replacement mutations in complementarity-determining regions in the inactive lambda 2-chain genes similar to the active lambda 1-chain genes suggested a mechanism that induces somatic mutation preferentially in this region even in the absence of antigenic selection.
Engineered secreted T-cell receptor alpha beta heterodimers.
Authors: Grégoire C , Rebaï N , Schweisguth F , Necker A , Mazza G , Auphan N , Millward A , Schmitt-Verhulst A M , Malissen B
Published: May 2006

We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface.
Molecular dynamics of dopamine at the D2 receptor.
Authors: Dahl S G , Edvardsen O , Sylte I
Published: May 2006

A three-dimensional model of the dopamine D2 receptor, assumed to be a target of antipsychotic drug action, was constructed from its amino acid sequence. The model was based on structural similarities within the super-family of guanine nucleotide-binding regulatory (G) protein-coupled neuroreceptors and has seven alpha-helical transmembrane segments that form a central core with a putative ligand-binding site. The space between two residues postulated to be involved in agonist binding, Asp-80 and Asn-390, perfectly accommodated an anti-dopamine molecule. Molecular electrostatic potentials were mainly negative on the synaptic side of the receptor model and around aspartate residues lining the central core and positive in the cytoplasmic domains. The docking of dopamine into a postulated binding site was examined by molecular dynamics simulation. The protonated amino group became oriented toward negatively charged aspartate residues in helix 2 and helix 3, whereas the dopamine molecule fluctuated rapidly between different anti and gauche conformations during the simulation. The receptor model suggests that protonated ligands are attracted to the binding site by electrostatic forces and that protonated agonists may induce conformational changes in the receptor, leading to G-protein activation, by increasing the electrostatic potentials near Asp-80.
Chemical attraction of kleptoparasitic flies to heteropteran insects caught by orb-weaving spiders.
Authors: Eisner T , Eisner M , Deyrup M
Published: May 2006

Insects of the heteropteran families Pentatomidae (stink bugs) and Coreidae (squash bugs), when being eaten by the orb-weaving spider Nephila clavipes, attract flies of the family Milichiidae. The flies aggregate on the bugs and, as kleptoparasites, share in the spider's meal. Stink bugs and squash bugs typically eject defensive sprays when attacked; they do so when caught by Nephila, but the spray only minimally affects the spider. Evidence is presented indicating that it is the spray of the bugs that attracts milichiids to the spider's catch.
Identification of Dictyostelium G alpha genes expressed during multicellular development.
Authors: Hadwiger J A , Wilkie T M , Strathmann M , Firtel R A
Published: May 2006

Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits.
Structural organization and transcription of the mouse gastric H+, K(+)-ATPase beta subunit gene.
Authors: Canfield V A , Levenson R
Published: May 2006

We have cloned and characterized the mouse gene encoding the beta subunit of H+, K(+)-ATPase (EC 3.6.1.36). The entire 10.5-kilobase transcription unit of the H+,K(+)-ATPase beta subunit gene was cloned in three overlapping cosmids encompassing approximately 46 kilobases of genomic DNA. A tight cluster of transcription initiation sites has been localized 24-25 nucleotides upstream of the translation start site and 28-29 nucleotides downstream of a TATA-like sequence. The H+, K(+)-ATPase beta subunit gene is split into seven exons encoding predicted structural domains of the beta subunit protein. The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons, and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the mouse H+,K(+)-ATPase beta subunit gene is identical to that of the mouse Na+,K(+)-ATPase beta 2 subunit gene. The conservation of genomic organization, together with the high sequence homology, indicates that the mouse H+,K(+)-ATPase beta and Na+,K(+)-ATPase beta 2 subunit genes originated from a common ancestral gene.
HLA-DR and -DQ gene polymorphism in West Africans is twice as extensive as in north European Caucasians: evolutionary implications.
Authors: Olerup O , Troye-Blomberg M , Schreuder G M , Riley E M
Published: May 2006

The HLA genes are the most polymorphic coding loci known in humans. DRB-DQA-DQB gene polymorphism was investigated by Taq I restriction fragment length polymorphism analysis in more than 700 West Africans and found to be almost twice as extensive in West Africans as in North European Caucasians. This finding indicates that Africans comprise the oldest and genetically most diverse human population and supports the hypothesis of the occurrence of a population bottleneck in the emergence of the White race. As in Caucasians, less than one-third of possible cis-encoded DQA-DQB combinations were encountered, indicating constraints on the pairing of DQ alpha and beta polypeptides. Heterozygote advantage (i.e., positive selection) was found for DRB, DQA, and DQB alleles as well as for DQA-DQB combinations. However, in West Africans as well as in North Europeans the observed frequencies of DRB-DQA-DQB homozygotes were close to neutrality expectations. Although the hypothesis that HLA polymorphism is maintained by parasite-driven overdominant selection is attractive, there is little evidence to support that view. We propose instead that one of the forces maintaining a low frequency of HLA homozygotes might be a decreased likelihood of potentially autoreactive T-cell clones escaping thymic selection in HLA heterozygotes. This would be consistent with the central role of HLA molecules as self/non-self discriminators.
Targeting of transgene expression to monocyte/macrophages by the gp91-phox promoter and consequent histiocytic malignancies.
Authors: Skalnik D G , Dorfman D M , Perkins A S , Jenkins N A , Copeland N G , Orkin S H
Published: May 2006

A component of a heterodimeric cytochrome b, designated gp91-phox, is required for the microbicidal activity of phagocytic cells and is expressed exclusively in differentiated myelomonocytic cells (granulocytes; monocyte/macrophages). In an attempt to identify cis-elements responsible for this restricted pattern of expression, we produced transgenic mice carrying reporter genes linked to the human gp91-phox promoter. Immunohistochemical and RNA analyses indicate that 450 base pairs of the proximal gp91-phox promoter is sufficient to target reporter expression to a subset of monocyte/macrophages. Mice expressing simian virus 40 large tumor antigen under control of the gp91-phox promoter develop monocyte/macrophage-derived malignancies with complete penetrance at 6-12 mo of age and provide an animal model of true histiocytic lymphoma. As these transgenes are inactive in most phagocytic cells that express the endogenous gp91-phox-encoding gene, we infer that additional genomic regulatory elements are necessary for appropriate targeting to the full complement of phagocytes in vivo.
Adrenocorticotropin receptors: functional expression from rat adrenal mRNA in Xenopus laevis oocytes.
Authors: Mertz L M , Catt K J
Published: May 2006

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A)+ RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. These were detected at 48 h and reached a maximum 72 h after microinjection of 20-40 ng of adrenal mRNA. In response to 1 microM ACTH, total cAMP production increased within 2.5 min and reached half-maximal and maximal levels (5-fold greater than basal) at 10 and 75 min, respectively, and then remained elevated for up to 5 h. Extracellular cAMP levels were much lower but showed prominent linear increases from almost undetectable levels, with 70- and 150-fold increases evident at 1 and 2 h, respectively. The half-maximal concentration (ED50) for stimulation of cAMP formation was 5 x 10(-8) M ACTH-(1-24); the ED50 for ACTH-(1-17) was 5 x 10(-7) M, and no response was observed with ACTH-(1-10). Size fractionation of rat adrenal poly(A)+ RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1- to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.
The T-cell-receptor repertoire in the synovial fluid of a patient with rheumatoid arthritis is polyclonal.
Authors: Uematsu Y , Wege H , Straus A , Ott M , Bannwarth W , Lanchbury J , Panayi G , Steinmetz M
Published: May 2006

We have analyzed the T-cell-receptor repertoire expressed in the synovial fluid of a patient with rheumatoid arthritis by using an inverse polymerase chain reaction. Total RNA was isolated from Ficoll-purified mononuclear cells and converted into circularized double-stranded cDNA. Specific amplification of alpha- and beta-chain variable regions (V alpha and V beta) was achieved with inverted alpha- and beta-chain constant region (C alpha and C beta) primer pairs, and the amplification products were cloned into phage vectors. A total of 78 alpha and 76 beta clones were sequenced, and 67 and 72 productively rearranged alpha and beta genes were identified, respectively. Thirty-one V alpha, 33 alpha-chain joining region (J alpha), 29 V beta, and 12 beta-chain joining region (J beta) gene segments were found in the productively rearranged clones, indicating that the T-cell repertoire expressed in the synovial fluid of this RA patient is highly heterogenous and polyclonal. Comparison of peripheral blood and synovial fluid repertoires showed that the most abundant V beta sequences, V beta 2.1 and V beta 3.1, were enriched in the inflamed joint by a factor of 2 to 3. It is possible that T cells expressing these V beta gene segments, which recognize bacterial superantigens, play a role in the disease.
Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6.
Authors: Rose T M , Bruce A G
Published: May 2006

Oncostatin M (OSM), a glycoprotein of Mr approximately 28,000 produced by activated monocyte and T-lymphocyte cell lines, was previously identified by its ability to inhibit the growth of cells from melanoma and other solid tumors. We have detected significant similarities in the primary amino acid sequences and predicted secondary structures of OSM, leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). Analysis of the genes encoding these proteins revealed a shared exon organization, suggesting evolutionary descent from a common ancestral gene. Using a panel of DNAs from somatic cell hybrids, we have shown that OSM, like LIF, is located on human chromosome 22. We have also demonstrated that OSM has the ability to inhibit the proliferation of murine M1 myeloid leukemic cells and can induce their differentiation into macrophage-like cells, a function shared by LIF, G-CSF, and IL-6. We propose that OSM, LIF, G-CSF, and IL-6 are structurally related members of a cytokine family that have in common the ability to modulate differentiation of a variety of cell types.
In vivo catalysis of a metabolically essential reaction by an antibody.
Authors: Tang Y , Hicks J B , Hilvert D
Published: May 2006

We have established a growth selection requirement for a catalytic antibody with modest chorismate mutase activity. Conversion of (-)-chorismate into prephenate is the key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. Strains of the yeast Saccharomyces cerevisiae containing an insertion mutation in the structural gene for the enzyme chorismate mutase (EC 5.4.99.5) require exogenous supplements of these two amino acids for efficient growth. Intracellular expression of the heterologous antibody catalyst in one such strain, identified by random mutagenesis and genetic selection, provides a substantial growth advantage under auxotrophic conditions; complementation was not observed with an unrelated esterolytic antibody. In addition to demonstrating that tailored immunoglobulin catalysts can carry out vital biochemical reactions in vivo, these experiments provide a powerful selection assay for identifying genetic changes within the antibody molecule itself that augment chemical efficiency.
Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.
Authors: French B T , Patrick D E , Grever M R , Trewyn R W
Published: May 2006

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.
Diets of yearling female primates (Papio cynocephalus) predict lifetime fitness.
Authors: Altmann S A
Published: May 2006

The foraging of yearling baboons (Papio cynocephalus) was studied in Amboseli National Park, Kenya. The nutrient content of the diets of individual females was compared with the composition of energy-maximizing optimal diets. Energy in the diets of all individuals fell appreciably short of their respective optima. Nonetheless, linear combinations of just two variables, protein intakes in excess of requirements and proximity of energy intakes to those specified in the optimal diets, were sufficient to provide good predictions of reproductive lifespans, numbers of infants and juveniles produced, and probability of surviving to adulthood.
Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing.
Authors: Kjems J , Garrett R A
Published: May 2006

The single 23S rRNA gene of the archaeon Staphylothermus marinus exhibits two introns which, at the RNA level, are located in highly conserved regions of domains IV and V. The RNA introns, which are 56 and 54 nucleotides long, respectively, can form single hairpin structures. In vivo, RNA splicing occurs efficiently, whereas in vitro pre-rRNA transcripts containing each intron were cleaved efficiently when incubated with archaeal cell extracts but were poorly ligated. The introns are cleaved by a mechanism which differs from the mechanisms of eukaryotic rRNA introns but resembles those of the rRNA intron of Desulfurococcus mobilis and the archaeal tRNA introns. The cleavage enzyme recognizes and cuts a putative bulge-helix-bulge structure that can form at the archaeal exon-intron junctions. Using a phylogenetic sequence comparison approach, we define the parts of this structural feature that are essential for cleavage. We also provide evidence for conformational changes occurring in the S. marinus 23S RNA, after cleavage, at both exon-exon junctions, which may account for the low yields of ligation observed in vitro.
2-nitro-3-(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane, two intermediates in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.
Authors: Halkier B A , Lykkesfeldt J , Møller B L
Published: May 2006

The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been studied in vitro by using [18O]oxygen and a microsomal enzyme system obtained from etiolated sorghum seedlings. The products formed were purified by HPLC and TLC, and the incorporation of [18O]oxygen was monitored by mass spectrometry. In the presence of NADPH and [18O]dioxygen, L-tyrosine is converted to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime with quantitative incorporation of an [18O]oxygen atom into the oxime function. The first step in this conversion is the N-hydroxylation of L-tyrosine to N-hydroxytyrosine. Administration of N-hydroxytyrosine as a substrate results in the production of 1-nitro-2-(p-hydroxyphenyl)ethane in addition to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, with quantitative incorporation of a single [18O]oxygen atom in all three products. These data demonstrate that the conversion of N-hydroxytyrosine to p-hydroxyphenylacetaldehyde oxime involves additional N-hydroxylation and N-oxidation reactions giving rise to the formation of 2-nitro-3-(p-hydroxyphenyl)propionate, which by decarboxylation produces aci-1-nitro-2-(p-hydroxyphenyl)ethane. Both compounds are additional intermediates in the pathway. The two [18O]oxygen atoms introduced by the N-hydroxylations are enzymatically distinguishable as demonstrated by the specific loss of the oxygen atom introduced by the first N-hydroxylation reaction in the subsequent conversion of aci-1-nitro-2-(p-hydroxyphenyl)ethane to (E)-p-hydroxyphenylacetaldehyde oxime. A high flux of intermediates through the microsomal enzyme system is obtained with N-hydroxytyrosine as a substrate. This renders the conversion of the aci-nitro compound rate limiting and results in its release from the active site of the enzyme system and accumulation of the tautomeric nitro compound.
Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice.
Authors: Yamada G , Ogawa M , Akagi K , Miyamoto H , Nakano N , Itoh S , Miyazaki J , Nishikawa S , Yamamura K , Taniguchi T
Published: May 2006

Interferons (IFNs) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation. In fact, IFNs inhibit growth of various normal and transformed cell types. Previously, a nuclear factor, IRF-1 (interferon regulatory factor 1), which binds to type I IFN and some IFN-inducible gene promoters, was identified and cloned. Since the IRF-1 gene is both virus and IFN inducible, an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation. In this study, we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer. In the transgenic mice, all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes (B cells). Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population. In fact, transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern.
Identification and characterization of "zinc-finger" domains by the polymerase chain reaction.
Authors: Pellegrino G R , Berg J M
Published: May 2006

We have developed a method for amplifying DNA fragments containing tandem arrays of "zinc-finger" sequences of the transcription factor IIIA (Cys2-His2) type by using the polymerase chain reaction. Because these sequences occur as tandem arrays, a ladder of bands is produced upon amplification using primers derived from the amino- and carboxyl-terminal sequences of a zinc-finger domain. The "rungs" of this ladder correspond to DNA fragments encoding one zinc-finger domain, two adjacent zinc-finger domains, and so on. This is demonstrated by isolating individual bands corresponding to n zinc-finger domains and reamplifying them with the same primers. This yields a band of the original size as well as bands corresponding to 1 through (n - 1) zinc-finger domains. Direct evidence that these bands encode zinc-finger domains was obtained by cloning and sequencing a collection of the amplification products. Due to the lack of redundancy in the sequences obtained, we conclude that each band corresponds to a large number of unique zinc-finger-encoding sequences. The results from amplification reaction mixtures using genomic DNA from a variety of sources as template provide further evidence that zinc-finger domains occur widely and frequently in eukaryotic genomes. We believe that this method is a powerful technique for the isolation and characterization of zinc-finger-encoding genes.
Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.
Authors: Wu Q , Li L , Cooper M D , Pierres M , Gorvel J P
Published: May 2006

The predicted amino acid sequence of the cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2]. In the present studies, we examined the enzymatic activity of this antigen. From brush border preparations of the small intestine, a rich source of many endopeptidases and exopeptidases, the BP-1 antibody selectively removed aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7] activity. The APA activity of a panel of cell lines correlated in linear fashion with cell-surface levels of the BP-1/6C3 antigen. APA activity was demonstrated for the BP-1/6C3 antigen immunopurified from the pre-B-cell membrane. This activity was enhanced by alkaline earth metals such as Ca2+ and was abrogated by amastatin and angiotensin, which are known competitive inhibitors of APA. The data indicate that the murine BP-1/6C3 antigen is active APA, an enzyme that catalyzes specifically the removal of unsubstituted, N-terminal glutamic acid and aspartic acid residues from peptides.
Distribution of glutamine and asparagine residues and their near neighbors in peptides and proteins.
Authors: Robinson A B , Robinson L R
Published: May 2006

In a statistical study of neighboring residues in 1465 peptides and proteins comprising 450,431 residues, it was found that the preferences for residues neighboring to glutamine and asparagine residues are consistent with the hypothesis that the rates of deamidation of these residues are of biological significance. Some dipeptide and tripeptide structures have special usefulness and some are especially undesirable. More such structures exist for amide residues than for other residues, and their specific types are those most relevant to the deamidation of amide residues under biological conditions.
Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.
Authors: Chappel M S , Isenman D E , Everett M , Xu Y Y , Dorrington K J , Klein M H
Published: May 2006

To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.
RNase-like domain in DNA-directed RNA polymerase II.
Authors: Shirai T , Go M
Published: May 2006

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.
Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli.
Authors: Kazakov S , Altman S
Published: May 2006

The location of phosphate residues involved in specific centers for binding of metal ions in M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was determined by analysis of induction of cleavage of RNA by metal ions. At pH 9.5, Mg2+ catalyzes cleavage of M1 RNA at five principal sites. Under certain conditions, Mn2+ and Ca2+ can each replace Mg2+ as the cofactor in the processing of precursor tRNAs by M1 RNA and P RNA, the RNA subunit of RNase P from Bacillus subtilis. These cations, as well as various metal ion inhibitors of the catalytic activity of M1 RNA, also promote cleavage of M1 RNA in a specific manner. Certain conditions that affect the catalytic activity of M1 RNA also alter the rate of metal ion-induced cleavage at the various sites. From these results and a comparison of cleavage of M1 RNA with that of a deletion mutant of M1 RNA and of P RNA, we have identified two different centers for binding of metal ions in M1 RNA that are important for the processing of the precursor to tRNA(Tyr) from E. coli. There is also a center for the binding of metal ions in the substrate, close to the site of cleavage by M1 RNA.
Nuclear scaffolds and scaffold-attachment regions in higher plants.
Authors: Hall G , Allen G C , Loer D S , Thompson W F , Spiker S
Published: May 2006

DNA in the nuclei of eukaryotic organisms undergoes a hierarchy of folding to be packaged into interphase and metaphase chromosomes. The first level of packaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber. Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs). Support for the ubiquitous nature of such higher order packaging of DNA is presented here by our work with plants. We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold. We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes. This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold. Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs. The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA fragments derived from the 3'-flanking regions of the root-specific genes are confirmed to contain SARs by this exogenous assay.
Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.
Authors: Morrison A , Bell J B , Kunkel T A , Sugino A
Published: May 2006

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.
Limitations in plasticity of the T-cell receptor repertoire.
Authors: Nanda N K , Apple R , Sercarz E
Published: May 2006

How constrained is T-cell recognition? Is a truncated T-cell receptor (TCR) repertoire, missing half of its V beta components (where V indicates variable), still broad enough to produce an antigen-specific T-cell response to all determinants? These questions can be answered for certain T-cell antigenic determinants whose response in the wild type is limited to specific gene segments. Our results show that mice with such a deletion in their TCR V beta genes (V beta truncated haplotype, Va beta) are unable to respond to two antigen determinants (sperm whale myoglobin 111-121/I-Ed and myelin basic protein 1-11/I-Au) whose response in the wild type is restricted to the missing V beta (V beta 8.2 in the case of 111-121/I-Ed and V beta 8.2 and V beta 13 in the case of 1-11/I-Au) gene segments. Fundamentally, this restriction could have been attributed to another aspect of immunodominance--that a favored TCR with high affinity would dominate the response, but in its absence, a hierarchy of T cells with lesser efficiency and expressing alternate TCR V genes could take over. However, from our experiments it has become evident that there is an absolute limit to the flexibility inherent in the TCR repertoire. Since it is clear that mouse populations have many ambient deletion ligands (such as self-superantigens) that can result in the loss of multiple V beta gene segments during normal T-cell development, these deletions can have serious consequences, such as unresponsiveness to the antigen as a whole--a hole in the repertoire--if a dominant determinant of that antigen normally shows restricted TCR V beta gene usage.
cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.
Authors: Parimoo S , Patanjali S R , Shukla H , Chaplin D D , Weissman S M
Published: May 2006

Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.
DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locus in yeast.
Authors: White M A , Wierdl M , Detloff P , Petes T D
Published: May 2006

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher rate of meiotic recombination than others. We show below that the binding of the RAP1 protein to a site upstream of the HIS4 gene is necessary for a high rate of meiotic (but not mitotic) recombination at this locus. A mutation in the RAP1 binding site at HIS4 results in a decrease in recombination; overproduction of RAP1 causes an increase in recombination at HIS4 above wild-type levels.
Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.
Authors: Fleming J G , Summers M D
Published: May 2006

The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus.
mRNA leader length and initiation codon context determine alternative AUG selection for the yeast gene MOD5.
Authors: Slusher L B , Gillman E C , Martin N C , Hopper A K
Published: May 2006

MOD5, a nuclear gene of Saccharomyces cerevisiae, encodes two isozymic forms of a tRNA-modification enzyme. These enzymes modify both cytoplasmic and mitochondrial tRNAs. Two inframe ATGs of the MOD5 gene are used for initiation of translation, and the form of the protein translated from the first AUG is imported into mitochondria. Protein translated from the second AUG functions in the cytoplasm. Since all transcripts contain both of these translational start sites and two proteins are made, the question arises as to the factors that influence the translation start-site choice. Extending the 5' ends of the MOD5 mRNA to include leader sequences of the ADH1 (alcohol dehydrogenase defective) transcript produces significant changes in the choice of AUGs. This suggests that for wild-type MOD5 transcripts, the length or structure of the leader sequence plays a role in AUG choice. The nucleotides surrounding the first ATG of MOD5 also have an effect on translation initiation. Altering these nucleotides changes initiation choice and suggests that ribosomal bypass of a suboptimal AUG is another mechanism controlling the alternate use of two initiation codons. Our data support the model that at least one MOD5 transcript is able to produce two proteins with different N-terminal sequences.
Excess nonsynonymous substitution of shared polymorphic sites among self-incompatibility alleles of Solanaceae.
Authors: Clark A G , Kao T H
Published: May 2006

The function of the self-incompatibility locus (S locus) of many plant species dictates that natural selection will favor high levels of protein diversity. Pairwise sequence comparisons between S alleles from four species of Solanaceae reveal remarkably high sequence diversity and evidence for shared polymorphism. The level of amino acid constraint was found to be significantly heterogeneous among different regions of the gene, with some regions being highly constrained and others appearing to be virtually unconstrained. In some regions of the protein, there was an excess of nonsynonymous over synonymous substitution, consistent with the strong diversifying selection that must operate on this locus. These hypervariable regions are candidates for the sites that determine functional allelic identity. Simple contingency table tests show that sites that have polymorphism shared between species have more nonsynonymous substitution than polymorphic sites that do not exhibit shared polymorphism. This is consistent with the idea that adaptive evolution favoring amino acid replacement is occurring at sites with shared polymorphism. Tests of clustered polymorphism reveal that an unusually low rate of recombination must be occurring in this locus, allowing very ancient alleles to preserve their identity.
Two-dimensional life?
Authors: de Duve Christian , Miller Stanley L.
Published: May 2006

A model [Wächtershäuser, G. (1988)
Microbiol. Rev.
52, 452-484], according to which life started in the form of a monomolecular layer of interacting anionic metabolites electrostatically bound to a positively charged surface, is examined critically. The model raises a number of thermodynamic and kinetic difficulties.
Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific.
Authors: Shuman S
Published: May 2006

Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.