bluuebunny/crossref_metadata_2025 huggingface dataset:
Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.
Authors: Mankin A S , Zyrianova I M , Kagramanova V K , Garrett R A
Published: May 2006

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.
Selective loss of a DNase I hypersensitive site upstream of the tyrosine aminotransferase gene in mice homozygous for lethal albino deletions.
Authors: Zaret K S , Milos P , Lia M , Bali D , Gluecksohn-Waelsch S
Published: May 2006

Several overlapping chromosomal deletions spanning the albino locus in the mouse cause perinatal lethality when homozygous and a block in the transcriptional induction of various unlinked hepatocyte-specific genes. Studies of such lethal albino deletion homozygotes in perinatal stages revealed a deficiency in the transcriptional inducibility of the tyrosine aminotransferase (TAT) gene by glucocorticoids; yet, glucocorticoid receptor and hormone levels were shown to be unaffected. To identify a molecular defect underlying the failure of inducible expression, we examined the chromatin structure of the TAT gene. Whereas in wild-type animals the TAT promoter becomes DNase I hypersensitive at birth, such hypersensitivity fails to develop in lethal albino deletion homozygotes. By contrast, the deletions do not affect the appearance of three DNase I-hypersensitive sites upstream of the TAT promoter in the liver, nor do they affect two hypersensitive sites upstream of the expressed alpha-fetoprotein gene. These findings demonstrate that the abnormality of chromatin structure identified in lethal albino deletion homozygotes occurs on a highly selective basis. Specifically, normal differentiation of the TAT promoter chromatin appears to depend directly or indirectly on the action and product of a gene mapping within the deleted region.
A cyclic peptide synthetase gene required for pathogenicity of the fungus Cochliobolus carbonum on maize.
Authors: Panaccione D G , Scott-Craig J S , Pocard J A , Walton J D
Published: May 2006

Specificity in many plant-pathogen interactions is determined by single genes in pathogen and host. The single locus for host-selective pathogenicity (TOX2) in the fungus Cochliobolus carbonum governs production of a cyclic tetrapeptide named HC-toxin. We have isolated a chromosomal region, 22 kilobases (kb) long, that contains a 15.7-kb open reading frame (HTS1) encoding a multifunctional cyclic peptide synthetase. The 22-kb chromosomal region is duplicated in toxin-producing isolates of the fungus but is completely absent from the genomes of toxin-nonproducing isolates. Mutants of the fungus with disruptions in both copies of HTS1, at either of two different sites within HTS1, were engineered by DNA-mediated transformation. Disruption of both copies at either site resulted in loss of ability to produce HC-toxin and loss of host-selective pathogenicity, but the mutants displayed different biochemical phenotypes depending on the site of disruption. The results demonstrate that TOX2 encodes, at least in part, a large, multifunctional biosynthetic enzyme and that the evolution of host range in C. carbonum involved the insertion or deletion of a large piece of chromosomal DNA.
Lymphoid cells transformed by Abelson virus require the v-abl protein-tyrosine kinase only during early G1.
Authors: Chen Y Y , Rosenberg N
Published: May 2006

Cells infected with temperature-sensitive transformation mutants of the Abelson murine leukemia virus express low levels of kinase activity at the nonpermissive temperature, causing transformed pre-B cells to die under these conditions. Examination of cell cycle profiles of such populations prior to cell death reveals that the cells accumulate in the G1 phase of the cell cycle. Following G1 arrest, the cells die via apoptosis, an active process of cell elimination. Cell synchronization and temperature-shift experiments show that G1 arrest reflects the requirement for a functional v-abl protein during early G1 and that the molecule is not required at other phases of the cell cycle. These data indicate that the substrate(s) critical to v-abl-mediated transformation is involved in regulating G1 transit and that these interactions are dominant over all other changes required for the multistep process that results in the fully malignant phenotype associated with v-abl expression in lymphoid cells.
gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex.
Authors: Orlando R A , Kerjaschki D , Kurihara H , Biemesderfer D , Farquhar M G
Published: May 2006

Using antibodies isolated from glomeruli of nephritic rats we have previously identified a 330-kDa cell surface glycoprotein (gp330) as a major pathogenic antigen of Heymann nephritis (HN), an experimental model of human membranous glomerulonephritis. Recently, we have isolated a cDNA clone, C14, encoding a polypeptide that contains a pathogenic epitope of HN responsible for the initiation of the disease. Subsequently, another protein, alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP), which is a subunit of the receptor for human alpha 2-macroglobulin/low density lipoprotein receptor-related protein (LRP), was shown to possess a high degree of sequence homology to the C14 protein (C14p). In this report, we have investigated the relationship between gp330, C14p, and alpha 2-MRAP. Immunoprecipitation studies demonstrate that gp330 forms a heterodimeric association with a 44-kDa polypeptide that is stable to detergent extraction and long-term centrifugation. Further, immunoblotting analysis on the purified complex indicates that the 44-kDa associated protein shares immunological identity to C14p and alpha 2-MRAP. In addition, antibodies eluted from glomeruli of HN rats and antibodies to a C14 fusion protein immunoprecipitated gp330 and the 44-kDa protein, demonstrating that the epitopes responsible for the initial events of HN are accessible within the complex. Based on these data, three models are proposed to explain how pathogenic epitopes in the gp330-44-kDa, HN antigenic complex may be presented at the cell surface and initiate the onset of HN.
Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.
Authors: Reddy S , Rayburn H , von Melchner H , Ruley H E
Published: May 2006

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.
At least three promoters direct expression of the mouse glucocorticoid receptor gene.
Authors: Strähle U , Schmidt A , Kelsey G , Stewart A F , Cole T J , Schmid W , Schütz G
Published: May 2006

We have characterized the gene for the mouse glucocorticoid receptor. The gene spans approximately 110 kilobases, and glucocorticoid receptor transcripts are assembled from nine exons. Expression of the gene is controlled by at least three promoters, resulting in glucocorticoid receptor transcripts with different 5' nontranslated exons. One promoter is cell-specific, found to be active only in T lymphocytes. The other two promoters are active to various degrees in all cell lines and tissues so far analyzed and are located in a CpG island. The promoter activities are accompanied by DNase I hypersensitivity sites in chromatin. In contrast to a conservation of exon-intron structure, differences in promoter organization suggest a divergence between the evolution of regulatory and coding regions among members of the steroid receptor super-family.
The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase.
Authors: Stepien P P , Margossian S P , Landsman D , Butow R A
Published: May 2006

Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclear-mitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G----T transversion that creates a Val----Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.
Evolution of the mouse t haplotype: recent and worldwide introgression to Mus musculus.
Authors: Morita T , Kubota H , Murata K , Nozaki M , Delarbre C , Willison K , Satta Y , Sakaizumi M , Takahata N , Gachelin G
Published: May 2006

Mouse t haplotypes are variants of chromosome 17, consisting of four inversions. Despite the homozygous lethality and pleiotropic effect on embryonic development, sperm production, and recombination, they have widely spread in natural populations of the house mouse (10-40% in frequency) because of the meiotic drive advantage. We sequenced 14 Tcp-1 (t-complex polypeptide 1) genes from four t haplotypes, nine wild mice, and a rat as a reference. From a comparison of intron sequences of 610 base pairs, we dated the origin of t haplotypes to 2.9 +/- 0.7 million years ago, which predates the splitting of Mus musculus subspecies (approximately 1 million years ago). However, the Tcp-1 intron sequences of t haplotypes from different M. musculus subspecies from various parts of the world show no divergence, indicating the recent introgression (no earlier than 0.8 million years ago) of a single ancestral type. Nucleotide changes in coding regions are also consistent with this conclusion. Hence, polymorphisms among t haplotypes including lethality factors have accumulated during this short time period independently in each M. musculus subspecies.
Tissue- and site-specific DNA recombination in transgenic mice.
Authors: Orban P C , Chui D , Marth J D
Published: May 2006

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries.
The budding yeast HRR25 gene product is a casein kinase I isoform.
Authors: DeMaggio A J , Lindberg R A , Hunter T , Hoekstra M F
Published: May 2006

The Saccharomyces cerevisiae HRR25 gene was identified as a regulator of DNA strand-break repair. HRR25 encodes a protein kinase that is closely related to bovine casein kinase I (CKI). CKI is a ubiquitous multipotential protein kinase. Rabbit polyclonal antibodies that recognize and immunoprecipitate Hrr25p have been generated and an immune complex protein kinase assay has been developed. The reaction depends upon HRR25 and shows that Hrr25p uses casein as a substrate. The identity between Hrr25p and bovine CKI suggests that Hrr25p is a yeast isoform of the CKI family and that CKIs may play a role in regulating DNA metabolism.
Functional analysis of the homeodomain-related proteins of the A alpha locus of Schizophyllum commune.
Authors: Specht C A , Stankis M M , Giasson L , Novotny C P , Ullrich R C
Published: May 2006

DNA-mediated transformation was used to correlate function with putative genes from three alternative A alpha mating-type loci (A alpha 1, A alpha 3, and A alpha 4) of Schizophyllum commune. Each DNA was tested in at least nine haploid strains, one for each of the nine A alpha mating types found in the world-wide population of S. commune. The Y and Z genes (tentatively identified by sequence analysis elsewhere) individually activate A alpha-regulated development when transformed into any strain with a different A alpha mating type. The only exceptions are when the Y alleles of A alpha 3 or A alpha 4 (i.e., Y3 or Y4, respectively) are introduced into an A alpha 1 strain (the A alpha 1 locus encodes Y1 but lacks a Z gene). These observations indicate that A alpha-regulated development is activated by the interaction (direct or indirect) of products from different genes (e.g., Z3 and Y1) rather than from different alleles of the same gene (e.g., Y1 and Y3). Therefore, the activating interaction is of the form ZiYj where i not equal to j and i and j are the A alpha mating types from which the Z and Y polypeptides, respectively, are derived. Transformations with truncated or mutagenized genes begin to define essential regions of the genes and their products. Activity is in some cases dependent upon the particular A alpha mating type of the recipient. A working hypothesis for the activation of A alpha-regulated development is proposed.
Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor.
Authors: Zhou Q Y , Li C , Olah M E , Johnson R A , Stiles G L , Civelli O
Published: May 2006

We have previously reported the selective amplification of several rat striatal cDNA sequences that encode guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. One of these sequences (R226) exhibited high sequence identity (58%) with the two previously cloned adenosine receptors. A full-length cDNA clone for R226 has been isolated from a rat brain cDNA library. The cDNA clone encodes a protein of 320 amino acids that can be organized into seven transmembrane stretches. R226 has been expressed in COS-7 and CHO cells and membranes from the transfected cells were screened with adenosine receptor radioligands. R226 could bind the nonselective adenosine agonist tritiated N-ethyladenosine 5'-uronic acid ([3H]NECA) and A1-selective agonist radioiodinated N6-2-(4-amino-3-iodophenyl)-ethyladenosine ([125I]APNEA) but not A1-selective antagonists tritiated 1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) and 8-(4-[([[(2-aminoethyl)amino]carbonyl]methyl)oxy]-phenyl)-1, 3-dipropylxanthine ([3H]XAC) or the A2-selective agonist ligands tritiated 2-[4-(2-carboxyethyl)phenyl]ethyl-amino 5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) and radioiodinated 2-[4-([2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl)phenyl]ethylamino 5'-N-ethylcarboxamidoadenosine. Extensive characterization with [125I]APNEA showed that R226 binds [125I]APNEA with high affinity (Kd = 15.5 +/- 2.4 nM) and the specific [125I]APNEA binding could be inhibited by adenosine ligands with a potency order of (R)-N6-phenyl-2-propyladenosine (R-PIA) = NECA greater than S-PIA greater than adenosine greater than ATP = ADP but not by antagonists XAC, isobutylmethylxanthine, and DPCPX. In R226 stably transfected CHO cells, adenosine agonists R-PIA, NECA, and CGS21680 inhibited by 40-50% the forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive G protein with an EC50 of 18 +/- 5.6 nM, 23 +/- 3.5 nM, and 144 +/- 34 nM, respectively. Based on these observations we conclude that R226 encodes an adenosine receptor with non-A1 and non-A2 specificity, and we thus name it the A3 adenosine receptor. mRNA analyses revealed that the highest expression of R226 was in the testis and low-level mRNAs were also found in the lung, kidneys, heart, and some parts of the central nervous system such as cortex, striatum, and olfactory bulb. The high-expression level of the A3 receptor in the testis suggests a possible role for adenosine in reproduction.
Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.
Authors: Ross S R , Choy L , Graves R A , Fox N , Solevjeva V , Klaus S , Ricquier D , Spiegelman B M
Published: May 2006

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.
Neurally evoked calcium transients in terminal Schwann cells at the neuromuscular junction.
Authors: Reist N E , Smith S J
Published: May 2006

We examined the effects of motor-nerve stimulation on the intracellular Ca2+ levels of Schwann cells, the glial cells at the frog neuromuscular junction. Schwann cells, which were loaded with the fluorescent Ca2+ indicator fluo-3 and examined by confocal microscopy, showed a transient increase in free Ca2+ within a few seconds of the onset of tetanic stimulation of the motor nerve. The Ca2+ response was specific to the synapse in that it was found in the terminal Schwann cells at the junction but not in the myelinating Schwann cells along the axon. The Ca2+ transients occurred in the presence of d-tubocurare, indicating that they were not mediated by nicotinic acetylcholine receptors and recurred when the stimulus was repeated. The Ca2+ response persisted after degeneration of the postsynaptic muscle fiber, demonstrating that the terminal Schwann cell was stimulated directly by presynaptic activity. The finding that terminal Schwann cells at the neuromuscular junction respond to presynaptic activity suggests that glial-cell function is modulated by synaptic transmission.
The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA.
Authors: Schleper C , Kubo K , Zillig W
Published: May 2006

The lemon-shaped "virus-like" particle SSV1 produced by the thermophilic archaeon Sulfolobus shibatae has not previously been observed to infect any host. Using a plaque assay suitable for the extreme growth conditions of this archaeon, we have shown infection of Sulfolobus solfataricus by SSV1. Upon infection, the viral genome was always found integrated into a tRNA gene of the host chromosome, a situation similar to that in S. shibatae, proving that site-specific integration is involved in establishing the lysogenic state. As in S. shibatae, UV-irradiation of lysogenized S. solfataricus led to virus production apparently not accompanied by cell lysis. We have also demonstrated the efficient uptake of exogenous DNA and its expression in Sulfolobus by transfecting S. solfataricus with SSV1 DNA by electroporation. Transfection efficiencies of up to 10(6) transfectants per microgram of DNA were obtained.
Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation.
Authors: Flanagan P M , Kelleher R J , Tschochner H , Sayre M H , Kornberg R D
Published: May 2006

Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators.
Origins of the Indo-Europeans: genetic evidence.
Authors: Sokal R R , Oden N L , Thomson B A
Published: May 2006

Two theories of the origins of the Indo-Europeans currently compete. M. Gimbutas believes that early Indo-Europeans entered southeastern Europe from the Pontic Steppes starting ca. 4500 B.C. and spread from there. C. Renfrew equates early Indo-Europeans with early farmers who entered southeastern Europe from Asia Minor ca. 7000 BC and spread through the continent. We tested genetic distance matrices for each of 25 systems in numerous Indo-European-speaking samples from Europe. To match each of these matrices, we created other distance matrices representing geography, language, time since origin of agriculture, Gimbutas' model, and Renfrew's model. The correlation between genetics and language is significant. Geography, when held constant, produces a markedly lower, yet still highly significant partial correlation between genetics and language, showing that more remains to be explained. However, none of the remaining three distances--time since origin of agriculture, Gimbutas' model, or Renfrew's model--reduces the partial correlation further. Thus, neither of the two theories appears able to explain the origin of the Indo-Europeans as gauged by the genetics-language correlation.
Derivation of a biologically contained replication system for human immunodeficiency virus type 1.
Authors: Chen H , Boyle T J , Malim M H , Cullen B R , Lyerly H K
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.
T-cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A.
Authors: Pontzer C H , Irwin M J , Gascoigne N R , Johnson H M
Published: May 2006

We have examined the interaction of the microbial superantigen staphylococcal enterotoxin A (SEA) with peptides corresponding to overlapping regions of the T-cell antigen receptor beta chain variable region V beta 3. SEA is known to stimulate murine T cells bearing certain V beta elements, among them V beta 3. Five peptides were synthesized representing amino acids 1-24, 20-44, 39-60, 57-77, and 74-95 of V beta 3. We demonstrate here that soluble V beta 3-bearing beta chains can bind to a complex of SEA and major histocompatibility complex class II and that the synthetic peptide V beta 3-(57-77) blocked this interaction. The peptide V beta 3-(57-77) also inhibited SEA-induced interferon-gamma production and SEA-induced proliferation of B10.BR spleen cells. Conversely, the peptide corresponding to amino acids 57-77 of V beta 8.2, a V beta element that is not recognized by SEA, decreased staphylococcal enterotoxin C-2-induced proliferation but did not affect SEA-induced proliferation. The peptide inhibition of SEA-induced function was due at least in part to inhibition of V beta 3-bearing T-cell activity, since the percentage of T cells reactive with an anti-V beta 3 monoclonal antibody was significantly reduced by V beta 3-(57-77). These data suggest that the region of V beta 3 encompassing amino acids 57-77 is an area that displays the appropriate sequence and conformation for binding of the SEA molecule and blocking of the resultant interaction with the T-cell antigen receptor.
Phosphorylation stimulates the transcriptional activity of the human beta 1 thyroid hormone nuclear receptor.
Authors: Lin K H , Ashizawa K , Cheng S Y
Published: May 2006

The role of phosphorylation on the gene activation activity of the human beta 1 thyroid hormone nuclear receptor (h-TR beta 1) was examined. h-TR beta 1 was found to be a phosphoprotein when expressed in COS-1 cells, with serine, threonine, and tyrosine (85:10:5) as the phosphorylation sites. Okadaic acid (a potent inhibitor of phosphatases 1 and 2A) at 0.1, 0.25, and 0.5 microM increased the phosphorylation of h-TR beta 1 by 3-, 7-, and 11-fold, respectively. The increase in phosphorylation was accompanied by a concomitant increase in phosphorylation was accompanied by a concomitant increase in receptor-mediated transcription in transient transfection assays. h-TR beta 1 purified from Escherichia coli was phosphorylated in vitro by the endogenous kinase from cellular extracts. Serine, threonine, and tyrosine were phosphorylated in a similar ratio to that found in COS-1 cells. The in vitro phosphorylation was stimulated by okadaic acid. Phosphorylation did not affect the binding of h-TR beta 1 to 3,3',5-triiodo-L-thyronine. However, phosphorylation of h-TR beta 1 resulted in an increase of its binding to DNA and conferred on it the ability to bind to nuclear accessory proteins. The results indicate that phosphorylation plays an important role in the transcriptional activity of h-TR beta 1.
Species specificity of renin kinetics in transgenic rats harboring the human renin and angiotensinogen genes.
Authors: Ganten D , Wagner J , Zeh K , Bader M , Michel J B , Paul M , Zimmermann F , Ruf P , Hilgenfeldt U , Ganten U
Published: May 2006

The renin-angiotensin system (RAS) is the most important regulatory system of electrolyte homeostasis and blood pressure. We report here the development of transgenic rats carrying the human angiotensinogen TGR-(hAOGEN) and human renin TGR(hREN) genes. The plasma levels and tissue distribution of the transcription and translation products from both genes are described. A unique species specificity of the enzyme kinetics was observed. The human RAS components in the transgenic rats did not interact with the endogenous rat RAS in vivo. Instead, infusions of exogenous human RAS components specifically interacted with human transgene translation products. Thus, infusion of human renin in TGR(hAOGEN) led to an increase of angiotensin II and an elevation of blood pressure, which could not be antagonized by the human-specific renin enzyme inhibitor Ro 42-5892. Rat renin also elevated blood pressure and angiotensin II in TGR(hAOGEN); however, this effect was not antagonized by the human renin inhibitor. Compared to mice, rats offer the advantage of chronic instrumentation and repetitive, sophisticated, hemodynamic, and endocrinological investigations. Thus, transgenic rat models with human-specific enzyme kinetics permit primate-specific analyses in non-primate in vivo and in vitro experimental systems.
Spontaneous malignant transformation of melanocytes explanted from Wf/Wf mice with a Kit kinase-domain mutation.
Authors: Larue L , Dougherty N , Porter S , Mintz B
Published: May 2006

The W/Kit mouse locus, affecting proliferation and survival of pigment cells, blood cells, and germ cells, is known to encode a tyrosine kinase growth factor receptor and is considered a protooncogene; yet it has not heretofore been causally implicated in any malignancies of those cells. The Wf/Wf mutant mouse coat comprises viable and inviable melanoblast clones, seen ultimately as pigmented and white transverse stripes--the latter more prominent. Judging from the pattern, all clones initially expand, and the inviable ones then undergo programmed cell death prenatally. To observe skin melanocytes of the viable clones during extended proliferation, the cells were explanted from individual young mice. An unusually large number of primary explants failed to survive--a result consistent with a growth handicap. In 3 of the 10 surviving cell lines, many cells spontaneously underwent a series of striking changes with the classic features of transformation. The two transformed lines that have been tested by grafting to immunosuppressed hosts formed undifferentiated invasive tumors compatible with malignant amelanotic melanoma. None of our 52 other melanocyte lines of the coisogenic wild-type strain and 13 other natural genotypes have become transformed under the same culture conditions. Molecular analysis of the Wf gene revealed a single change from wild-type: a point mutation affecting the catalytic region in the kinase domain of the Kit protein. The apparent growth disadvantage due to the mutation may allow selection for melanocytes mobilizing more efficient pathways, thus leading to neoplasia. Production of both viable and inviable melanoblast clones is unlikely to be due only to the kinase mutation; possibly the degree, duration, and consistency of expression of this locus may be controlled by cis elements outside the coding region.
Hydroxyl radical cleavage of tRNA in the ribosomal P site.
Authors: Hüttenhofer A , Noller H F
Published: May 2006

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.
Multicellular oxidant defense in unicellular organisms.
Authors: Ma M , Eaton J W
Published: May 2006

Although catalase is thought to be a major defense against hydrogen peroxide (H2O2), the catalase activity within individual Escherichia coli fails to protect against exogenous H2O2. Contrary to earlier reports, we find that dilute suspensions of wild-type and catalase-deficient E. coli are identical in their sensitivity to H2O2, perhaps because even wild-type, catalase-positive E. coli cannot maintain an internal/external concentration gradient of this highly diffusible oxidant. However, concentrated suspensions or colonies of catalase-positive E. coli do preferentially survive H2O2 challenge and can even cross-protect adjacent catalase-deficient organisms. Furthermore, high-density catalase-positive--but not catalase-negative--E. coli can survive and multiply in the presence of competitive, peroxide-generating streptococci. These observations support the concept that bacterial catalase may defend colonial, but not individual, E. coli against environmental H2O2. Group protection by the activity of enzymes that mitigate oxidative stress may have been a driving force in the evolution of multicellular organisms.
CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1.
Authors: Hague B F , Sawasdikosol S , Brown T J , Lee K , Recker D P , Kindt T J
Published: May 2006

Human CD4 (HuCD4) is the principal receptor for human immunodeficiency virus type 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.
Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.
Authors: Van Dyke B R , Bakan D A , Glover K A , Hegenauer J C , Saltman P , Springer B A , Sligar S G
Published: May 2006

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.
Hydrolysis and reconjugation of gibberellin A20 glucosyl ester by seedlings of Zea mays L.
Authors: Schneider G , Jensen E , Spray C R , Phinney B O
Published: May 2006

The [6-2H]glucosyl ester of [17-13C,3H]gibberellin A20 (GA20) was injected into light-grown 14-day-old seedlings of normal, dwarf-1, and dwarf-5 maize (Zea mays L.). The plant material was extracted 24 h later, and the extracts were purified by solvent partitioning, column chromatography, and HPLC. 13C-labeled metabolites were identified from the purified extracts by full-scan gas chromatography/mass spectrometry and selected ion current monitoring in conjunction with Kovats retention indices. The metabolites, [13C]GA20, [13C]GA29, [13C]GA20-13-O-glucoside, and [13C]GA29-2-O-glucoside, were identified from normal, dwarf-1, and dwarf-5 seedlings. [13C]GA8 and [13C]GA8-2-O-glucoside were also identified from normal and dwarf-5 seedlings but not from dwarf-1 seedlings. The data provide definitive evidence for the endogenous hydrolysis by the seedlings of the introduced conjugate and its reconjugation to three glucosides.
Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related.
Authors: Büttner M , Xie D L , Nelson H , Pinther W , Hauska G , Nelson N
Published: May 2006

Oxygenic photosynthesis of chloroplasts and cyanobacteria involves two photosystems, which originate from different prokaryotic ancestors. The reaction center of photo-system 2 (PS2) is related to the well-characterized reaction center of purple bacteria, while the reaction center of photosystem 1 (PS1) is related to the green sulfur bacteria, as is convincingly documented here. An operon encoding the P840 reaction center of Chlorobium limicola f.sp. thiosulfatophilum has been cloned and sequenced. It contains two structural genes, coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the PS1 reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS1, and for the acceptors A0, A1, and FeS center X are conserved. The second protein is related to the PS1 subunit carrying the FeS centers A and B. An adjacent third gene, not belonging to the reaction center, encodes a protein related to dolichyl-phosphate-D-mannose synthase from yeast. The different origins of PS1 and PS2 are discussed.
Sequence and expression of a membrane-associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120.
Authors: Curtis B M , Scharnowske S , Watson A J
Published: May 2006

The binding of the human immunodeficiency virus (HIV) envelope glycoprotein gp120 to the cell surface receptor CD4 has been considered a primary determinant of viral tropism. A number of cell types, however, can be infected by the virus, or bind gp120, in the absence of CD4 expression. Human placenta was identified as a tissue that binds gp120 in a CD4-independent manner. A placental cDNA library was screened by expression cloning and a cDNA (clone 11) encoding a gp120-binding protein unrelated to CD4 was isolated. The 1.3-kilobase cDNA predicts a protein of 404 amino acids with a calculated M(r) of 45,775 and organized into three domains: an N-terminal cytoplasmic and hydrophobic region, a set of seven complete and one incomplete tandem repeat, and a C-terminal domain with homology to C-type (calcium-dependent) lectins. A type II membrane orientation (N-terminal cytoplasmic) is predicted both by the cDNA sequence and by the reactivity of C-terminal peptide-specific antiserum with the surface of clone 11 transfected cells. Native and recombinant gp120 and whole virus bind transfected cells. gp120 binding is high affinity (kd, 1.3-1.6 nM) and inhibited by mannan, D-mannose, and L-fucose; once bound, gp120 is internalized rapidly. Collectively, these data demonstrate that the gp120-binding protein is a membrane-associated mannose-binding lectin. Proteins of this type may play an important role in the CD4-independent association of HIV with cells.
Hierarchical analysis of linkage disequilibrium in Rhizobium populations: evidence for sex?
Authors: Souza V , Nguyen T T , Hudson R R , Piñero D , Lenski R E
Published: May 2006

Many bacterial species exhibit strong linkage disequilibrium of their chromosomal genes, which apparently indicates restricted recombination between alleles at different loci. The extent to which restricted recombination reflects limited migration between geographically isolated populations versus infrequent mixis of genotypes within populations is more difficult to determine. We examined the genetic structure of Rhizobium leguminosarum biovar phaseoli populations associated with wild and cultivated beans (Phaseolus spp.) over several spatial scales, ranging from individual host plants to throughout the Western Hemisphere. We observed significant linkage disequilibrium at scales at least as small as a cultivated plot. However, the amount of disequilibrium was much greater among isolates collected throughout the Western Hemisphere than among isolates from one area of Mexico, even when disequilibrium was quantified using an index that scales for allelic diversity. This finding suggests that limited migration between populations contributes substantially to linkage disequilibrium in Rhizobium. We also compared the genetic structure for R. leguminosarum bv. phaseoli taken from a cultivated plot with that for Escherichia coli obtained from one human host in an earlier study. Even at this fine scale, linkage disequilibrium in E. coli was very near the theoretical maximum level, whereas it was much less extreme in the local population of Rhizobium. Thus, the genetic structure for R. leguminosarum bv. phaseoli does not exclude the possibility of frequent mixis within local populations.
Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis.
Authors: Ambudkar S V , Lelong I H , Zhang J , Cardarelli C O , Gottesman M M , Pastan I
Published: May 2006

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).
Primary structure of tektin A1: comparison with intermediate-filament proteins and a model for its association with tubulin.
Authors: Norrander J M , Amos L A , Linck R W
Published: May 2006

Tektins are proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to those of intermediate-filament proteins. We report here the sequence of a cDNA for tektin A1, one of the main tektins from Strongylocentrotus purpuratus sea urchin embryos. By hybridization analysis, tektin A mRNA appears maximally at ciliogenesis. The predicted structure of tektin A1 (M(r) 52,955) is a series of alpha-helical rod segments separated by nonhelical linkers. The two halves of the rod appear homologous and are probably related by gene duplication. Comparison of tektin A1 with intermediate-filament proteins, including nuclear lamins, reveals a low amino acid homology but similar molecular motif, i.e., pattern of helical and nonhelical domains. This study indicates that tektins are unique proteins but may be evolutionarily related to intermediate-filament proteins, and suggests a structural basis for the interaction of tektins and tubulin in microtubules.