Single Specific Site Research Articles

Isolation and Mapping of Human T-Cell Protein Tyrosine Phosphatase Sequences: Localization of Genes and Pseudogenes Discriminated Using Fluorescence Hybridization with Genomic versus cDNA Probes

This work reports the isolation, partial characterization, and chromosomal mapping of several human T-cell protein tyrosine phosphatase (PTPase) sequences and provides a direct comparison of the specificity of cDNA versus genomic probes in discriminating the location of genes versus pseudogenes by fluorescence in situ hybridization. In initial attempts to map the T-cell (TC) PTP gene using a 2-kb cDNA, several labeled sites were noted, raising the possibility of multiple related sequences within the genome. To address this, four genomic clones were obtained with homology to the TC PTP cDNA and characterized for their primary structure and their position within the human genome. Based on the presence or absence of an open reading frame and the intron/exon structure, two of these clones were found to be overlapping sequences encoding the true TC PTP gene and two were highly related but distinct processed pseudogenes. The TC PTP gene (gene symbol PTPN2) encoded by clones L17-2 and L5-1 localized to chromosome 18p11.2-p11.3, whereas pseudogenes encoded by clone L17-1, entitled TCPS1 (gene symbol PTPN2P1), and clone L18, entitled TCPS13 (gene symbol PTPN2P2), mapped to chromosomes 1q22-q24 and 13q12-q13, respectively. A direct comparison of the specificity of genomic and cDNA probes demonstrated that under identical conditions the genomic probes (containing both exon and intron sequences) readily identified a single specific site of hybridization, whereas the cDNA identified sites of both the gene and its pseudogenes. While providing mapping and sequencing information on the TC PTPase sequences, this work illustrates a strategy for addressing a recurrent problem in gene mapping studies where highly related sequences exist within the genome.

Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation.

C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase.

Tn7: a target site-specific transposon.

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.

Sequence requirements for DNA rearrangements induced by the terminal repeat of herpes simplex virus type 1 KOS DNA

We investigated the sequence requirements for the site-specific DNA cleavages and recombinational genome isomerization events driven by the terminal repeat or a sequence of herpes simplex virus type 1 KOS DNA by inserting a series of mutated a sequences into the thymidine kinase locus in the intact viral genome. Our results indicate that sequences located at both extremities of the a sequence contribute to these events. Deletions entering from the Ub side of the a sequence progressively reduced the frequency of DNA rearrangements, and further deletion of the internal DR2 repeat array had an additional inhibitory effect. This deletion series allowed us to map the pac1 site-specific DNA cleavage signal specifying the S-terminal cleavage to a sequence that is conserved among herpesvirus genomes. Constructs lacking this signal were unable to directly specify the S-terminal cleavage event but retained a reduced ability to give rise to S termini following recombination with intact a sequences. Deletions entering from the Uc side demonstrated that the copy of direct repeat 1 located adjacent to the Uc region plays an important role in the DNA rearrangements induced by the a sequence: mutants lacking this sequence displayed a reduced frequency of novel terminal and recombinational inversion fragments, and further deletions of the Uc region had a relatively minor additional effect. By using a construct in which site-specific cleavage was directed to heterologous DNA sequences, we found that the recombination events leading to genome segment inversion did not occur at the sites of DNA cleavage used by the cleavage-packaging machinery. This observation, coupled with the finding that completely nonoverlapping portions of the a sequence retained detectable recombinational activity, suggests that inter-a recombination does not occur by cleavage-ligation at a single specific site in herpes simplex virus type 1 strain KOS. The mutational sensitivity of the extremities of the a sequence leads us to hypothesize that the site-specific DNA breaks induced by the cleavage-packaging system stimulate the initiation of recombination.

Molecular epidemiology of macrolides-lincosamides-streptogramin B resistance in Staphylococcus aureus and coagulase-negative staphylococci.

Macrolides-lincosamides-streptogramin B (MLS) resistance is commonly found in Staphylococcus aureus and coagulase-negative staphylococci (22 and 45%, respectively, among isolates from three New Jersey hospitals). We have examined representative subsets of 107 MLS-resistant isolates for the molecular nature of the resistance determinant, the erm gene, by dot blot and Southern hybridization analysis. All of 35 S. aureus isolates examined and 39 of 42 coagulase-negative isolates examined were found to harbor the ermA or ermC evolutionary variant. Genes of the ermC class occurred exclusively on a small plasmid similar to or indistinguishable from one (pNE131) previously described in S. epidermidis. Genes of the ermA class occurred exclusively in the chromosome, and restriction patterns indicated that they were part of a transposon, Tn554, characteristic of the classical S. aureus ermA strain. Unlike S. aureus ermA strains examined previously, which harbor Tn554 at a single specific (primary) site, four of our S. aureus isolates had second inserts at different chromosomal sites. The majority of our coagulase-negative isolates had two or more inserts, neither of which occurred at the classical primary site and many of which differed from one another in location (as inferred from restriction patterns). Coagulase-negative staphylococci constitute a large reservoir of the ermA and ermC class of determinants, with clear potential for interspecies spread.

Nucleoside transporter of cerebral microvessels and choroid plexus.

The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1-0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguanosine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleosides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood-brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of approximately 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.

125I-spiperone: A novel ligand for D 2 dopamine receptors

125I-Spiperone binds with high affinity (K D 0.3 nM) to a single specific site (B max 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40–60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (K i's 1–10 nM). Selective dopamine antagonists such as sulpiride (K i 50 nM) are dopamine agonists such as apomorphine (K i 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D 2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D 2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo , using single photon tomography.

Multiple Destination Trip Bias in Recreation Benefit Estimation

Balanced and multiple use of the public lands has become the theme of much of federal land use policy.1 As additional and alternative uses of the public lands are proposed, the measurement of the value of publicly managed recreation facilities is necessary in order to make rational comparisons. For the most part these alternative uses have quantifiable benefits and costs determined through market operations. As a matter of social policy, however, access to many publicly managed recreation facilities is often provided at prices less than the market prices that would otherwise prevail. Since prices determined in a market economy usually provide the basis for calculating the benefits and costs of various commodities or activities, situations involving commodities or activities acquired without the use of market exchange pose certain difficulties for value estimation. It is necessary in these cases to derive visitor willingness-to-pay from observed nonmarket data or from direct consumer responses to questions about recreation values. One widely accepted technique for estimating the willingness-to-pay for a recreation site in cost-benefit studies is the Clawson-Knetsch travel cost method.2 Recently published regulations implementing revised Principles and Standards for Planning Water and Related Land Resources will result in increased application of this technique in federal planning. The advantages and disadvantages of using the travel cost method for estimating recreation benefits have been widely discussed (e.g., Pearce [1978]). The travel cost method assumes, among other things, that all travel costs are incurred exclusively to obtain access to the single specific recreation site being valued and thus differences in travel cost may be treated as a proxy for price. This study (1) examines the consequences of multiple destination trips on travel cost estimates of benefits, (2) suggests a means of correcting the bias that arises under such circumstances, and (3) obtains nearly identical value estimates from the travel cost method and from a direct question on willingness-to-pay. The magnitude of multiple destination bias is

Viral DNA sequences in adenovirus-transformed cells.

Nononcogenic adenovirus 2 (Ad2) and adenovirus 5 (Ad5) are able to transform rodent cells in vitro (Freeman et al. 1967; Williams 1973). Graham et al. (1975) showed that only the left end of the adenoviral genome is necessary for in vitro transformation. However, it appeared that in a number of transformed cell lines additional segments of adenoviral DNA were present (Sambrook et al. 1975b). This observation rendered it rather improbable that a single specific site on the adenoviral genome is involved in the process of integration. The occurrence of different segments of the viral DNA in the transformed cell raises several questions: (1) Are there preferred sites of integration on the host and viral genomes? (2) Are the host and viral sequences involved in integration homologous? (3) How does the pattern of integration of viral sequences vary in different transformed cell lines? (4) Are the patterns of integration stable during...

The DNa-protein relaxation complex of the plasmid RK2: Location of the site-specific nick in the region of the proposed origin of transfer

The broad jost range plasmid, RK2, has been isolated as a DNA-protein relaxation complex. Nicking of the plasmid DNA in the relaxation complex occurs at a single specific site (rlx) located approximately 20 kb away from the origin of DNA replication. A cis-acting function required for plasmid transfer, the presumptive origin of transfer, maps in the same region as rlx. The region of RK2 encompassing rlx has been cloned onto pBR322 and shown to promote mobilization of the hybrid plasmid by an RK2 derivative. These results indicate that the RK2 relaxation complex nicks at or near the origin of transfer of the RK2 plasmid.

LHRH‐PITUITARY PLASMA MEMBRANE BINDING: THE PRESENCE OF SPECIFIC BINDING SITES IN OTHER TISSUES

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II ( Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence, ▪ with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.

Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase.

A new adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of DPNH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3'-FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (Ki equals 1.0 times 10-4 M) prior to the irreversible modification. Protection against modification by 3'-FSBA is provided by ADP and by high concentrations of DPNH, but not by the inhibitor GTP, the substrate alpha-keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3'-tfsba. the modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of DPNH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent tdpnh inhibitory site. It is proposed that the regulatory sites for ADP and DPNH are partially overlapping and that 3'FSBA functions as a specific affinity label for the DPNH inhibitory site of glutamate dehydrogenase. It is anticipated that 3'-p-fluorosulfonylbenzolyadenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.

Analysis of the RNA of defective VSV particles

Viral mRNA isolated from infected cells and the virion RNA isolated from two classes of defective interfering particles have been analyzed by RNA-RNA duplex reactions. The results show that the RNA of both defective interfering particles is viral, not host in origin. The RNA isolated from the two defective particles represents homogeneous populations of molecules containing only part of the genetic information present in the whole VSV genome. Annealing competition experiments indicate that if any overlap exists between the two, it is less than 220 nucleotides. We conclude from the data presented that a rudimentary physical map of the VSV and DI particle genomes is ▪ Our results suggest that there is not a single specific site that is required for autointerference.

Specific initiation site for simian virus 40 deoxyribonucleic acid replication.

Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with (3)H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication.

Further studies on loss of T-antigen from somatic hybrids between mouse cells and SV40-transformed human cells.

The study of hybrids from three crosses between mouse cells and SV40-transformed human cells have established a positive correlation between the loss of human chromosomes and that of the SV40-induced T-antigen from the hybrid cells. These results, as well as those of other workers, provide strong support for the hypothesis of the integration of the SV40 genome in the chromosomes of transformed cells. Further, it has been shown that hybrid cells which have lost T-antigen are capable of synthesizing this antigen upon infection with SV40, thereby demonstrating that loss of the viral antigen from the hybrid cells is not due to loss of some cellular gene required for the expression of the viral genome. Results of karyological analyses of the hybrid cells argue against the existence of a single specific integration site for the SV40 genome in human cells.

The Influence of Deuterium Oxide and Organic Solvents on the Interaction of Respiratory Chain Components

Abstract 1. The influence of D2O and organic solvents on the rate of oxidation of added substrates by heart muscle particles and tightly coupled rat liver mitochondria has been investigated. Solvents examined include glycerol, sucrose, dimethyl sulfoxide, dimethyl formamide, and ethylene glycol. Each of these solvents was found to induce a marked inhibition of respiratory chain activity. 2. The relative constancy in the steady state reduction level of the respiratory carriers at each increment of solvent concentration prevents the assignment of a single specific site of respiratory inhibition, and favors the hypothesis that the solvent effects are due to a nonspecific inhibition which operates in a similar manner at several sites in the respiratory chain. 3. No correlation was found between the inhibition of respiratory activity by solvents and the viscosity of the solvent reaction mixtures. 4. The organic solvent inhibition of the DPNH oxidase activity of heart muscle particles was a linear function of the water content of the solvent reaction mixtures in which the particles were suspended, and independent of the concentration, tonicity, or chemical nature of the solvent used. 5. It is suggested that several protonolytic reactions may be operative during the transfer of reducing equivalents from substrate to oxygen by way of the respiratory chain. The respiratory chain components mediating these reactions are in some manner accessible to, and markedly influenced by, the aqueous phase surrounding the particles, and therefore do not function entirely in a lipid nonaqueous milieu.