bluuebunny/crossref_metadata_2025 huggingface dataset:
An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.
Authors: Graves M C , Lim J J , Heimer E P , Kramer R A
Published: May 2006

In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation.
Sequence comparisons of retroviral proteins: relative rates of change and general phylogeny.
Authors: McClure M A , Johnson M S , Feng D F , Doolittle R F
Published: May 2006

The inferred amino acid sequences of 10 specific gene products from nine retroviruses were aligned by computer, all evolutionary distances between them calculated, and evolutionary trees constructed. Not unexpectedly, the various gene products are changing at different rates, the reverse transcriptase being the least and the envelope proteins the most different from one retrovirus to another. For the most part, trees based on the retroviral enzyme sequences are congruent, indicating that extensive genetic recombination has not been a major factor in the evolution of the central part of the genome. In the case of envelope protein sequences, however, the sequences clearly exhibit evidence of multiple cross-over events between quite distantly related retroviruses. A composite phylogenetic tree was constructed from the four retroviral enzyme sequences, and a number of important historical happenings were interpreted in the light of the time scale it affords.
Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.
Authors: Wold M S , Kelly T
Published: May 2006

The replication of simian virus 40 (SV40) DNA is largely dependent upon cellular replication proteins. To define these proteins we have made use of a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication. Systematic fractionation-reconstitution experiments indicate that there are a minimum of six cellular proteins that are required for efficient viral DNA replication in vitro. We report here the purification of one of these proteins, replication protein A (RP-A), to homogeneity. RP-A is a multisubunit protein that contains four tightly associated polypeptides of 70, 53, 32, and 14 kDa. Partial proteolysis experiments indicate that the 53-kDa polypeptide is closely related to the 70-kDa polypeptide, suggesting that it may be a proteolytic fragment of the larger subunit. RP-A is absolutely required for reconstitution of SV40 DNA replication in vitro. The purified protein binds to single-stranded DNA and is required for the large tumor (T)-antigen-mediated unwinding of DNA molecules containing the SV40 origin of DNA replication. These properties are consistent with the possibility that RP-A plays a central role in the generation of a single-stranded region at the origin prior to initiation of DNA synthesis. The protein may also function to facilitate unwinding of the parental DNA strands during the elongation phase of SV40 DNA replication.
Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.
Authors: Benko D M , Haltiwanger R S , Hart G W , Gibson W
Published: May 2006

A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine.
Chromosomal organization of the cytochrome P450-2C gene family in the mouse: a locus associated with constitutive aryl hydrocarbon hydroxylase.
Authors: Meehan R R , Speed R M , Gosden J R , Rout D , Hutton J J , Taylor B A , Hilkens J , Kroezen V , Hilgers J , Adesnik M
Published: May 2006

Cytochromes P-450 represent a superfamily of enzymes with a central role in the metabolism of drugs, chemical toxins, and carcinogens. We have used genetic analysis to establish the complexity and catalytic function of a recently identified constitutively expressed murine hepatic cytochrome P-450 encoded by P450-2C. Southern blotting analysis shows that there are at least seven or eight genes within this family in the mouse and rat and that DNA restriction fragment length variants between different mouse inbred strains are observed. Analysis of recombinant inbred strains derived from these parent strains shows (i) these genes are clustered within 1 centimorgan, (ii) this gene family does not correspond to any of the known cytochrome P-450 loci or map near any well-characterized genomic markers, and (iii) this gene family segregates to within 1-2 centimorgans of a locus controlling constitutive aryl hydrocarbon hydroxylase activity in mice. With use of Chinese hamster/mouse somatic cell hybrids, the P450-2C locus was assigned to a region of mouse chromosome 19 that appears to be syntenic with the previously mapped human P450C2C locus on human chromosome 10. By in situ hybridization to mitotic mouse chromosomes, we have localized this region to the tip of chromosome 19. These results are discussed in relation to the physiological roles of this P-450 family in foreign compound metabolism and steroid oxidations.
Mitochondrial DNA rearrangement associated with fertility restoration and cytoplasmic reversion to fertility in cytoplasmic male sterile
Phaseolus vulgaris
L
Authors: Mackenzie S. A. , Pring D. R. , Bassett M. J. , Chase C. D.
Published: May 2006

Restoration of pollen fertility to cytoplasmic male sterile (CMS)
Phaseolus vulgaris
by a nuclear restorer gene provides a system for studying nuclear-cytoplasmic interactions. Introduction of a nuclear restorer gene to this CMS line of
P. vulgaris
(CMS-Sprite) results in a mitochondrial genome rearrangement similar to that observed upon spontaneous cytoplasmic reversion to fertility. Three spontaneous heritable cytoplasmic revertants were derived from CMS-Sprite. Five fully fertile restored lines were also produced by using restorer line R-351 (BC
3
F
3
populations). Comparison of the mitochondrial DNA restriction patterns of CMS-Sprite, the three fertile revertants, and the five restored lines revealed loss of a 6.0-kilobase (kb)
Pst
I fragment in all restored and revertant lines. Southern hybridizations with a 1.3-kb
Bam
HI clone, internal to the 6.0-kb
Pst
I fragment, as a probe revealed two configurations of 6.0-kb homologous sequences in the sterile cytoplasm; one of the configurations was lost upon reversion or restoration. Mitochondrial DNA rearrangement has thus been observed upon restoration by a nuclear restorer gene in this CMS system.
Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene.
Authors: Millán J L , Manes T
Published: May 2006

A full-length placental alkaline phosphatase (PLAP) cDNA was used to identify and clone the PLAP-like Nagao isozyme gene from human genomic libraries. The entire nucleotide sequence of the gene reveals the existence of 11 exons interrupted by 10 small introns (76-427 base pairs). Putative regulatory sequences have been identified in the promoter regions as well as dispersed in the introns. The deduced amino acid sequence of the Nagao isozyme indicates that the mature molecule is composed of 513 amino acids, of which 12 residues are different from the sequence of PLAP (98% homology). A sequence derived from exon III of the Nagao isozyme gene was used to synthesize a peptide (NH2-Lys-Leu-Gly-Pro-Glu-Thr-Phe-Leu-Ala-COOH) that contains two mutations with respect to the corresponding PLAP sequence. This peptide elicited rabbit polyclonal antibodies that reacted specifically with the seminoma Nagao isozyme but not with PLAP in electrophoretic transfer blots. These results indicate that the tumor, and possibly the normal testis, Nagao isozyme is encoded by a gene referred to as germ-cell alkaline phosphatase gene that differs from the PLAP gene expressed by syncitiotrophoblastic cells.
Temporal regulation in development: negative and positive cis regulators dictate the precise timing of expression of a Drosophila chorion gene.
Authors: Mariani B D , Lingappa J R , Kafatos F C
Published: May 2006

We have used germ-line transformation to dissect the cis regulatory elements responsible for the transcriptional control of an internally marked Drosophila chorion gene (s15-P) during development. A 73-base-pair segment of the proximal 5'-flanking DNA contains sequences essential for the tissue-specific expression and the precise "late" temporal regulation of that gene. A substitute s36-1 segment of similar location can provide the tissue-specific function and imparts an early temporal regulation characteristic of gene s36-1. Within the regulatory DNA of s15-P, at least three adjacent elements are recognizable: an essential operationally positive element (TCACGT) that is shared by s36-1 and other chorion genes, irrespective of temporal specificity; a second positive element that is required for the normal late expression of s15-P; and, farthest upstream, a negative element that represses precocious expression during the early choriogenic stages.
Recombination of haplotypes leads to biased estimates of admixture proportions in human populations.
Authors: Chakraborty R , Smouse P E
Published: May 2006

A population formed by genetic admixture of two or more source populations may exhibit considerable linkage disequilibrium between genetic loci. In the presence of recombination, this linkage disequilibrium declines with time, a fact that is often ignored when considering haplotypes of closely linked systems [e.g., Gm serum group (gamma globulins), HLA, and, more recently, restriction fragment length polymorphisms]. Recombination alters haplotype frequencies over time, and the haplotype-derived measures of admixture proportions from haplotype frequencies in generations following the admixture event become progressively more biased. The direction and extent of this bias can be predicted only when the history of admixture is known. Numerical illustration suggests that this bias is problematic whenever rt greater than 0.05, where r is the recombination rate between linked loci and t is the time (in generations) that has elapsed since the admixture event. In general, even the haplotype frequencies defined by multiple restriction fragment length polymorphism should be used with caution for admixture analysis. When recombination rates or the time since admixture are not precisely known, it is advantageous to consider each restriction fragment length polymorphism site separately for admixture analysis.
Multiple regulatory regions on the 5' side of the mouse E alpha gene.
Authors: Thanos D , Mavrothalassitis G , Papamatheakis J
Published: May 2006

The function of the 5'-flanking region of the mouse major histocompatibility complex gene Ed alpha has been studied by deletion analysis with the chloramphenicol acetyltransferase gene as a transient expression marker in various cell lines. This analysis reveals the presence of several control regions on the 5' side of the gene. Sequences between base pair (bp) -873 and bp -353 have a negative function in human and mouse fibroblasts but not in the mouse macrophage line WEHI-3. Additional positive and negative elements have been mapped between bp -353 and bp -38. A gamma-interferon response region has been also identified within that sequence. the 5' and 3' boundaries of the gamma-interferon response region have been located between bp -164 and bp -43. Inducible human cell lines showed the same gamma-interferon response region endpoints with the mouse cell line WEHI-3. A DNA fragment spanning the equivalent region of the mouse Ed beta gene confers gamma-interferon inducibility to the simian virus 40 and alpha-globin promoters in an orientation-independent manner. We further provide evidence that the conserved sequence motifs on the 5' side of all major histocompatibility complex class II genes are indispensable for gamma-interferon induction.
Qa-region class I gene expression: identification of a second class I gene, Q9, encoding a Qa-2 polypeptide.
Authors: Soloski M J , Hood L , Stroynowski I
Published: May 2006

A feature of the expression of the tissue-specific class I antigen Qa-2 is the quantitative variation among mouse strains. Recently, the class I gene Q7 has been shown to encode a protein product that is biochemically indistinguishable from the lymphocyte-bound Qa-2 molecule. Utilizing gene transfection, we have identified a second Qa-2 subregion class I gene (Q9), in H-2b mice, which encodes a polypeptide biochemically similar to the Q7 and the Qa-2 polypeptides. Furthermore, we have observed that cell lines transfected with the allelic forms of the Q7 gene from C57BL/10 (Qa-2hi) or BALB/C (Qa-2low) display quantitative differences in cell-surface expression. Based on these studies, we suggest that gene dosage and allele-specific variation in cell-surface expression contribute to the strain-specific variation in the levels of Qa-2 antigen expression.
Multiple endocrine neoplasia induced by the promiscuous expression of a viral oncogene.
Authors: Reynolds R K , Hoekzema G S , Vogel J , Hinrichs S H , Jay G
Published: May 2006

There is increasing evidence for the importance of events that govern and influence the interaction between the transformed cell and its host being ultimately responsible for the establishment of the cancer phenotype. To derive an animal model that will allow us to define some of these phenomena at the molecular level, we have chosen to induce the expression of a viral oncogene in all tissue types, with the hope of identifying sites that are more susceptible to malignant transformation. When the gene for simian virus 40 large tumor antigen (T antigen) was placed under the control of a major histocompatibility complex class I gene enhancer, the resulting transgenic mice not only developed choroid plexus papillomas, as seen with wild-type simian virus 40, but also lymphoid hyperplasia and multiple endocrine neoplasias. The development of lymphoid hyperplasia was preceded by an elevated level of expression of T antigen in these tissues at an early age. Surprisingly, the striking thymic hyperplasia has not been observed to progress toward malignancy. The multiple endocrine neoplasias developed later in life and involved the pancreas, pituitary, thyroid, adrenals, and testes. While not preceded by an elevated level of expression of T antigen, once endocrine tumors appeared they quickly progressed toward malignant growth. Although other tissues also exhibited a basal level of expression of the viral oncogene similar to that detected in endocrine tissues, they rarely developed tumors. This transgenic mouse model seems particularly suitable for a molecular understanding of events responsible for certain tissue types being so much more susceptible to neoplastic conversion, with others being so refractory.
Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone.
Authors: Hanson S R , Harker L A
Published: May 2006

Since the antithrombin action of heparin fails to interrupt arterial thrombosis, a mediating role for thrombin (EC 3.4.21.5) in the formation of high-shear platelet-dependent thrombus has been unproven. To determine whether thrombin is important in acute arterial thrombus formation and to assess the therapeutic potential of inhibiting its action, the effects of the synthetic covalent antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl) on arterial-flow vascular graft thrombosis and occlusion have been studied in a nonhuman primate model. Continuous intravenous infusion of FPRCH2Cl (100 nmol/kg per min) into vascular graft-bearing baboons (Papio anubis) abolished (i) vascular graft 111In-platelet deposition, (ii) vascular graft occlusion, (iii) thrombus-associated in vivo release of platelet-specific proteins and fibrinopeptides, (iv) platelet hemostatic plug formation, (v) thrombin-induced platelet aggregation ex vivo, and (vi) thrombin-induced blood clotting. The effects of FPRCH2Cl largely disappeared within 15 min after the infusion had been discontinued. FPRCH2Cl produced no detectable cardiovascular or other acute side effects. In contrast, sustained comparably anticoagulating levels of heparin had no effect upon 111In-platelet graft deposition, graft occlusion, platelet function as measured by the bleeding time, platelet aggregation ex vivo, or release of platelet-specific proteins in vivo. We conclude that thrombin is the principal mediator of platelet-dependent hemostatic plug formation and of the formation of platelet-dependent high-flow acute graft thrombosis and occlusion. Moreover, FPRCH2Cl or other synthetic antithrombins may provide effective antithrombotic therapy for both arterial and venous thrombosis by simultaneously inhibiting platelet activation and fibrin formation.
Patch-clamp recording of amino acid-activated responses in "organotypic" slice cultures.
Authors: Llano I , Marty A , Johnson J W , Ascher P , Gähwiler B H
Published: May 2006

Patch-clamp recording techniques were used to study the properties of amino acid-activated channels in cultured "organotypic" slices from rat cerebellum and hippocampus. Hippocampal pyramidal cells responded to the three main glutamatergic agonists, N-methyl-D-aspartate (N-Me-D-Asp), quisqualate, and kainate, whereas Purkinje cells responded only to quisqualate and kainate. Analysis of single-channel events recorded in outside-out patches from hippocampal neurons showed large conductance events (50 pS), which occurred more frequently in the presence of glycine. These events could be produced by N-Me-D-Asp and also, at low frequency, by quisqualate. On the other hand, 50-pS events were never observed in Purkinje neurons. This supports the hypothesis that N-Me-D-Asp and "non-N-Me-D-Asp" receptors are distinct molecular entities. Comparison of whole-cell and outside-out patch recordings from Purkinje cells revealed a clear spatial segregation of gamma-aminobutyric acid (GABA) and glutamate receptors: although GABA receptors are found at high density in somatic membrane, quisqualate and kainate receptors are mostly extrasomatic. The results show that organotypic slice cultures are amenable to patch-clamp methods. They also show that, in these cultures, amino acids receptors have specific distribution patterns according to cell type and to region within a cell.
Macromolecular organization of human centromeric regions reveals high-frequency, polymorphic macro DNA repeats.
Authors: Jabs E W , Goble C A , Cutting G R
Published: May 2006

To analyze the macromolecular organization of human centromeric regions, we used alpha-satellite, or alphoid, repetitive DNA sequences specific to the centromeres of human chromosomes 6 (D6Z1), X (XC), and Y (YC-2) and the technique of pulsed-field gel electrophoresis. Genomic DNA from 24 normal, unrelated individuals was digested and separated into fragments ranging from 23 kilobases (kb) to 2 megabases (Mb) in length. Digestion with 12 different restriction enzymes with 4- to 8-base-pair recognition sequences and hybridization with alphoid sequences revealed chromosome-specific hybridization patterns. Similarities in the organization of the centromeric regions of the three chromosomes included NotI, SfiI, and SalI fragments of greater than 2 Mb and Sau3A1 and Alu I fragments of less than 150 kb. Each restriction enzyme with a 6-base-pair recognition sequence (Ava II, BamHI, HindIII, Hpa I, Pst I, Sal I, Sst I, and Xba I) detected polymorphic DNA fragments of 50 kb to 2 Mb. Forty percent or more of the individuals screened revealed a unique hybridization pattern with these enzymes and at least one of the three chromosome-specific alphoid probes. Five individuals differed from one another in hybridization pattern for each of the three enzymes HindIII, HpaI, and SstI and for each of the three centromeric probes. All 24 individuals could be distinguished on the basis of unique hybridization patterns with only two enzymes and one chromosome-specific alphoid probe. Family studies showed that these polymorphisms are inherited. The high frequency of these macro restriction fragment length polymorphisms illustrates the high degree of variability of the centromeric region among normal individuals and demonstrates its usefulness for DNA fingerprinting and pericentromeric mapping by linkage analysis.
High-level erythroid expression of human alpha-globin genes in transgenic mice.
Authors: Ryan T M , Behringer R R , Townes T M , Palmiter R D , Brinster R L
Published: May 2006

The human alpha 1-globin gene was fused downstream of two erythroid-specific DNase I super-hypersensitive sites that are normally located upstream of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and expression was analyzed in 16-day fetal livers and brains. All 11 fetuses that contained intact copies of the transgene expressed correctly initiated human alpha-globin mRNA in the erythroid fetal liver but not in brain. Levels of expression ranged from 4% to 337% of endogenous mouse beta-globin mRNA. A human alpha-globin construct that did not contain super-hypersensitive sites was not expressed. These results demonstrate that human beta-globin locus activation sequences can stimulate high levels of human alpha-globin gene expression in erythroid tissue of transgenic mice. The results also provide a foundation for experiments designed to coexpress human alpha- and beta-globin genes in transgenic mice and suggest a feasible approach for production of a mouse model for human sickle cell disease.
Sex- and hormone-dependent antigen immunoreactivity in developing rat hypothalamus.
Authors: Tobet S A , Fox T O
Published: May 2006

Morphological sex differences in adults can result from differential gonadal steroid exposure during critical perinatal periods. This study describes the use of a monoclonal antibody we have developed to study mechanisms of sexual differentiation of brain structure and function. Used as a marker in immunocytochemistry, antibody AB-2 revealed subsets of cells, including radial glia, transiently during the perinatal period. Peak reactivity in radial glia was on embryonic day 19 in males and on postnatal day 1 in females. On postnatal day 1, AB-2 immunoreactivity in radial glia was 2-fold greater in females than in males. Greater activity was detected in males on one side of the brain than the other (2- to 4-fold, depending on the region). To test the hormone dependence of this sex difference, pregnant rats were injected with testosterone propionate to expose fetal females to androgen on embryonic day 18. This resulted in lower levels of AB-2 immunoreactivity in radial glia of the treated female offspring on postnatal day 1 relative to control females, and the pattern was bilaterally asymmetric, approaching that of males. Thus the difference between sexes in immunoreactivity with AB-2 as a marker was hormone dependent in a predictable manner. Whether this marker is revealing a sex difference in accessibility of antigen by immunocytochemistry or a sex difference in intrinsic antigen levels is not yet resolved. In either case these results support the hypothesis that certain hormone-dependent molecular events occur transiently during development.
Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences.
Authors: Huibregtse J M , Engelke D R , Thiele D J
Published: May 2006

Copper-inducible transcription of the yeast metallothionein gene (CUP1) occurs by means of cis-acting upstream activation sequences (UAS) and trans-acting cellular factors. We have used a high-resolution chromosomal footprinting technique to detect the interaction of cellular factors with UASCUP1. Our results demonstrate that one or more cellular factors bind to UASCUP1 in a copper-inducible fashion. This copper-inducible binding is enhanced in a yeast strain that harbors several copies of the positive regulatory gene ACE1 and is not detectable in yeast cells that contain a nonfunctional (ace1-delta 1) locus. The correlation between yeast metallothionein gene activation and copper-inducible occupation of UASCUP1 sequences suggests that the binding of metallothionein transcriptional regulatory factors to cis-acting control sequences is copper-inducible.
Theory of hexagonal and stripe phases in monolayers
Authors: McConnell Harden M.
Published: May 2006

Epifluorescence microscopy can be used to visualize the shapes of solid lipid domains in two-phase regions of monolayers at the air—water interface. The shapes of certain lipid domains result from a competition between a one-dimensional line tension and long-range intermolecular electrostatic repulsion. Under specified conditions, a finite two-dimensional domain with one shape can undergo a sharp transition to a second shape, as the area of the domain is changed. Two-dimensional infinite arrays of domains can also have transitions involving changes in the shapes and patterns of domains, such as the stripe to hexagonal phase transition [Andelman, D., Brochard, F. & Joanny, J. F. (1987)
J. Chem. Phys.
86, 3673-3681]. The present paper treats the hexagonal and stripe phases with the same approximations and methods of calculation as used previously for the isolated, finite domains. It is shown that one effect of electrostatic repulsion between domains is to cause these domains to increase in size as they approach one another on monolayer compression. It is also shown that there can be two distinct conditions where the hexagonal and stripe phases coexist.
Characterization of oligonucleotide transport into living cells.
Authors: Loke S L , Stein C A , Zhang X H , Mori K , Nakanishi M , Subasinghe C , Cohen J S , Neckers L M
Published: May 2006

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptor-mediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.
Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.
Authors: Domier L L , Franklin K M , Hunt A G , Rhoads R E , Shaw J G
Published: May 2006

Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters. The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail. Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA. Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG). Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.
Landmark mapping: a general method for localizing cysteine residues within a protein.
Authors: Nefsky B , Bretscher A
Published: May 2006

We describe a general method to locate the positions of cysteine residues relative to the amino terminus of a protein, using a modified chemical cleavage of the polypeptide backbone at cysteine. The cleavage reaction introduces the carbon atom of 14CN into the carboxyl-terminal fragment produced at each cleavage of the polypeptide chain. Peptides containing the amino terminus of the intact protein are not labeled; all other peptides are labeled at their amino termini. Partial cleavage of a protein followed by gel electrophoresis and autoradiography identifies a ladder of unlabeled peptides that maps positions of the cysteine residues relative to the protein amino terminus. To map individual proteins present in a complex mixture, the polypeptides are cyanolated in solution with 14CN, and the modified proteins are separated by discontinuous SDS/PAGE. The gel is stained, and the desired protein is excised, cleaved at cysteine within the gel slice, and mapped in the second dimension by gel electrophoresis. These techniques are demonstrated with proteins of known sequence containing from zero to five cysteine residues. The cysteine "landmark map" should be particularly useful in locating protein modifications, in questions of protein similarity, and in mapping functional domains. A strategy is also presented for locating other residues in the polypeptide, for which specific cleavage methods exist.
Direct activation of cGMP-dependent channels of retinal rods by the cGMP phosphodiesterase.
Authors: Bennett N , Ildefonse M , Crouzy S , Chapron Y , Clerc A
Published: May 2006

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).
Chlorambucil effectively induces deletion mutations in mouse germ cells.
Authors: Russell L B , Hunsicker P R , Cacheiro N L , Bangham J W , Russell W L , Shelby M D
Published: May 2006

The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated post-stem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with approximately 1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stage-response pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germ-line mutations at highest frequencies, chlorambucil may be the mutagen of choice.
Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA.
Authors: Bishop D K , Andersen J , Kolodner R D
Published: May 2006

A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae. The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA. Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene. The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon. The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction. With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested. A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA. T/C was repaired with an intermediate efficiency. The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions. Strains carrying the repair-deficient pms1-1 mutation were also tested. The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells. In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.
The current source of human Alu retroposons is a conserved gene shared with Old World monkey.
Authors: Britten R J , Stout D B , Davidson E H
Published: May 2006

A significant fraction of human Alu repeated sequences are members of the precise, recently inserted class. A cloned member of this class has been used as a probe for interspecies hybridization and thermal stability determination. The probe was reassociated with human, mandrill, and spider monkey DNA under conditions such that only almost perfectly matching duplexes could form. Equally precise hybrids were formed with human and mandrill DNA (Old World monkey) but not with spider monkey DNA (New World). These measurements as well as reassociation kinetics show the presence in mandrill DNA of many precise class Alu sequences that are very similar or identical in quantity and sequence to those in human DNA. Human and mandrill are moderately distant species with a single-copy DNA divergence of about 6%. Nevertheless, their recently inserted Alu sequences arise by retroposition of transcripts of source genes with nearly identical sequences. Apparently a gene present in our common ancestor at the time of branching was inherited and highly conserved in sequence in both the lineage of Old World monkeys and the lineage of apes and man.
A synthetic peptide defines a serologic IgA response to a human papillomavirus-encoded nuclear antigen expressed in virus-carrying cervical neoplasia.
Authors: Dillner J , Dillner L , Robb J , Willems J , Jones I , Lancaster W , Smith R , Lerner R
Published: May 2006

The growing awareness of the role of human papillomavirus (HPV) in cervical carcinoma has triggered a search for uncomplicated detection methods. To define a serologic response to HPV, we synthesized peptides based on sequences deduced from the genome of HPV type 16, the most common malignancy-associated type of HPV. One of these peptides reacted with IgA antibodies present in sera from 24 of 33 patients with cervical intraepithelial neoplasia or cervical carcinoma, whereas this peptide reacted with only 6 of 27 sera from individuals without cervical intraepithelial neoplasia. Immunoaffinity-purified human antipeptide IgA antibodies detected HPV-specific 58- and 48-kDa proteins in cervical carcinoma cell extracts and also detected a nuclear antigen in HPV-carrying cervical cancer cell lines and cervical intraepithelial neoplasia biopsied tissue. These antigens were also detected with mouse monoclonal and rabbit polyclonal antibodies to the same peptide. The results indicate that screening for infection with malignancy-associated types of HPV may be possible by simple synthetic peptide-based serology.
Parvalbumin increases in the caudate putamen of rats with vitamin D hypervitaminosis.
Authors: de Viragh P A , Haglid K G , Celio M R
Published: May 2006

The influence of chronic vitamin D3 application on the concentration of the four calcium-binding proteins parvalbumin, the 28-kDa calbindin-D, calmodulin, and S-100 was studied in various brain regions and in the kidney. Young rats were administered daily 20,000 international units of vitamin D3 per kg (body weight) over a period of 4 months. This chronic treatment resulted in a clinically mild hypervitaminosis that did not affect the content of calmodulin, the 28-kDa calbindin-D, and S-100. Also the concentration of parvalbumin in the cerebral cortex, hippocampus, and kidney remained unchanged. On the other hand, parvalbumin was increased about 50% in the caudate putamen of hypervitaminotic animals as compared to controls. Our results indicate that the metabolism of parvalbumin in the caudate putamen can be influenced by variations of the blood level of this steroid hormone.
A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication.
Authors: Akiyama M , Maki H , Sekiguchi M , Horiuchi T
Published: May 2006

Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.
Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.
Authors: Sladek F M , Melian A , Howard-Flanders P
Published: May 2006

4,5',8-Trimethylpsoralen (psoralen) plus near UV light produces interstrand crosslinks and monoadducts in DNA, both of which are mutagenic. In Escherichia coli, crosslinks are incised by UvrABC excinuclease, an event that can lead to homologous recombination and repair. To determine whether UvrABC incision of crosslinks is a step in the path to mutagenesis as well as repair, the effect of DNA homologous to a target gene on a plasmid was determined. pSV2-gpt DNA was treated with psoralen and transformed into a pair of hosts: one was gpt+, the other was delta (gpt-lac)5. The DNA was extracted and transformed into a tester strain [delta (gpt-lac)5] in which Gpt- mutations in the plasmid were scored. The results show that psoralen-induced mutations were reduced to background levels by the presence of the gpt+ homolog in the host chromosome. delta gpt hosts that were constitutively induced for the SOS response yielded point mutations, whereas noninduced hosts yielded almost exclusively large deletions. Since crosslinks were estimated to be responsible for most of the mutations observed, we conclude that the premutagenic lesion of psoralen crosslinks is recombinagenic and therefore very likely to be the product of UvrABC incision.
The chloroplast genome exists in multimeric forms
Authors: Deng Xing-Wang , Wing Rod A. , Gruissem Wilhelm
Published: May 2006

Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. We found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density. DNA gyrase decatenation analysis demonstrates that the majority of the DNA molecules are oligomers rather than catenanes. The relative amounts of monomer, dimer, trimer, and tetramer forms, quantitated by molecular hybridization, are 1, 1/3, 1/9, and 1/27, respectively, and do not change during leaf maturation. The possible mechanisms of chloroplast DNA oligomer formation are discussed.
Mouse/human chimeric monoclonal antibody in man: kinetics and immune response.
Authors: LoBuglio A F , Wheeler R H , Trang J , Haynes A , Rogers K , Harvey E B , Sun L , Ghrayeb J , Khazaeli M B
Published: May 2006

A mouse/human chimeric monoclonal antibody (mAb) composed of the variable regions of murine 17-1A mAb and the constant regions of human IgG-1K immunoglobulin was administered to 10 patients with metastatic colon cancer. Four patients received single infusion of 10 mg (n = 2) or 40 mg (n = 2). Six patients received three infusion of 10 mg (n = 3) or 40 mg (n = 3) at 2-week intervals. The pharmacokinetics were similar at both dose levels and at the second and third infusions. The plasma disappearance curves were best fit by a two-compartment model having a mean alpha T1/2 of 17.5 hr (range 13-23 hr) and a mean beta T1/2 of 100.5 hr (range 65-139 hr). One patient who received three 40-mg doses of chimeric IgG-1K 17-1A mAb (day 0, 14, and 28) was the only patient to exhibit a detectable but modest antibody reactivity to chimera on days 63 and 84. The antibody reactivity was inhibited by murine 17-1A mAb, indicating that the antibody response was directed to the murine variable region of the chimera. The patients had no toxic or allergic reactions. This chimeric form of 17-1A mAb has an approximate 6-fold longer circulation time and appears to be substantially less immunogenic than its murine counterpart. These characteristics may provide an advantage in the clinical application of such chimeric molecules in therapeutic trials in humans.
Molecular cloning of cDNA encoding Sm autoantigen: derivation of a cDNA for a B polypeptide of the U series of small nuclear ribonucleoprotein particles.
Authors: Ohosone Y , Mimori T , Griffith A , Akizuki M , Homma M , Craft J , Hardin J A
Published: May 2006

The Sm snRNPs play a central role in the processing of pre-mRNA. Anti-Sm antibodies, the diagnostic hallmark of systemic lupus erythematosus, target the B'/B and D polypeptides of these snRNPs. We have used patient autoantibodies to clone a cDNA from a human fibroblast cDNA library that encodes the full length of a polypeptide identical with, or closely related to, polypeptide B. This cDNA is comprised of 1139 bases and contains an open reading frame of 855 nucleotides that is capable of encoding 285 amino acids. The first 223 amino acids at the NH2 terminus exhibit nearly complete homology with polypeptide N, a newly recognized brain- and heart-specific component of Sm snRNPs. The derived amino acid sequence for B differs from that of the N polypeptide primarily by a 50-amino acid insert 12 residues upstream from the homologous COOH termini of these polypeptides. The structural differences in these cDNAs for B and N may regulate tissue-specific alternative splicing mechanisms for mRNA. In addition, these clones make it possible to map in fine detail the most characteristic autoimmune responses of systemic lupus erythematosus.
Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells.
Authors: Tateno M , Gonzalez-Scarano F , Levy J A
Published: May 2006

Certain human immunodeficiency virus (HIV) strains were shown to infect human fibroblastoid cells. Replication was demonstrated only by coculturing normal peripheral blood mononuclear cells or human T-lymphotropic virus type 1-transformed T-cell lines with the infected human cells. This infection of human fibroblastoid cells did not involve the CD4 molecule and did not have the properties of endocytosis. Human sera could be distinguished by their ability to neutralize HIV infection of the fibroblastoid versus human T-cell lines. These observations demonstrate further that other mechanisms for viral entry, besides CD4 binding, must be considered for HIV. They also indicate the wide cellular host range and heterogeneity of HIV strains. The possibility that fibroblastoid cells serve as a reservoir for the AIDS virus and are involved in connective tissue disorders of infected individuals merits attention.