bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for linkage of the apolipoprotein A-II locus to plasma apolipoprotein A-II and free fatty acid levels in mice and humans.
Authors: Warden C H , Daluiski A , Bu X , Purcell-Huynh D A , De Meester C , Shieh B H , Puppione D L , Gray R M , Reaven G M , Chen Y D
Published: May 2006

Although it has been hypothesized that the synteny between mouse and human genes provides an approach to the localization of genes that determine quantitative traits in humans, this has yet to be demonstrated. We tested this approach with two quantitative traits, plasma apolipoprotein A-II (apoAII) and free fatty acid (FFA) levels. ApoAII is the second most abundant protein of high density lipoprotein particles, but its function remains largely unknown. We now show that, in a backcross between strains Mus spretus and C57BL/6J, apoAII levels correlate with plasma FFA concentrations on both chow (P < 0.0001) and high-fat (P < 0.0003) diets and that apoAII levels are linked to the apoAII gene (P < 0.0002). To test whether variations of the apoAII gene influence plasma lipid metabolism in humans, we studied 306 individuals in 25 families enriched for coronary artery disease. The segregation of the apoAII gene was followed by using an informative simple sequence repeat in the second intron of the gene and two nearby genetic markers. Robust sib-pair linkage analysis was performed on members of these families using the SAGE linkage programs. The results suggest linkage between the human apoAII gene and a gene controlling plasma apoAII levels (P = 0.03). Plasma apoAII levels were also significantly correlated with plasma FFA levels (P = 0.007). Moreover, the apoAII gene exhibited linkage with a gene controlling FFA levels (P = 0.003). Evidence for nonrandom segregation was seen with markers as far as 6-12 centimorgans from the apoAII structural locus. These data provide evidence, in two species, that the apoAII gene is linked to a gene that controls plasma apoAII levels and that apoAII influences, by an unknown mechanism, plasma FFA levels. The results illustrate the utility of animal studies for analysis of complex traits.
Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.
Authors: Cantor G H , McElwain T F , Birkebak T A , Palmer G H
Published: May 2006

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.
"BFA bodies": a subcompartment of the endoplasmic reticulum.
Authors: Orci L , Perrelet A , Ravazzola M , Wieland F T , Schekman R , Rothman J E
Published: May 2006

A specialized region of the endoplasmic reticulum--the BFA body--is defined by the site of accumulation of coatomer when nonclathrin coat protein (COP)-coated vesicle assembly is prevented by the drug brefeldin A (BFA). BFA bodies are formed by part smooth, part rough domains of endoplasmic reticulum that are cis to the classical transitional endoplasmic reticulum and to BFA-induced Golgi remnants.
Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein.
Authors: Inoue S , Orimo A , Hosoi T , Kondo S , Toyoshima H , Kondo T , Ikegami A , Ouchi Y , Orimo H , Muramatsu M
Published: May 2006

Estrogen receptor (ER)-binding fragments were isolated from human genomic DNA by using a recombinant ER protein. Using one of these fragments as a probe, we have identified an estrogen-responsive gene that encodes a putative zinc finger protein. It has a RING finger motif present in a family of apparent DNA-binding proteins and is designated estrogen-responsive finger protein (efp). efp cDNA contains a consensus estrogen-responsive element at the 3' untranslated region that can act as a downstream estrogen-dependent enhancer. Moreover, efp is regulated by estrogen as demonstrated at both the mRNA and the protein level in ER-positive cells derived from mammary gland. These data suggest that efp may represent an estrogen-responsive transcription factor that mediates phenotypic expression of the diverse estrogen action. Thus, the genomic binding-site cloning may be applicable for isolation of the target genes of other transcription factors.
Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain.
Authors: Baskaran R , Dahmus M E , Wang J Y
Published: May 2006

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Phosphorylation of the CTD occurs during formation of the initiation complex and is correlated with the transition from complex assembly to elongation. Previously, serine and threonine residues within the CTD have been shown to be modified by the addition of phosphate and by the addition of O-linked GlcNAc. Our results establish that the CTD is also modified in vivo by phosphorylation on tyrosine. Furthermore, a nuclear tyrosine kinase encoded by the c-abl protooncogene phosphorylates the CTD to a high stoichiometry in vitro. Under conditions of maximum phosphorylation, approximately 30 mol of phosphate are incorporated per mol of CTD. The observation that the CTD is not phosphorylated by c-Src tyrosine kinase under identical conditions indicates that the CTD is not a substrate of all tyrosine kinases. Phosphorylation of tyrosine residues within the CTD may modulate the interaction of RNA polymerase II with the preinitiation complex and, hence, may be important in regulating gene expression.
The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators.
Authors: Dorn R , Krauss V , Reuter G , Saumweber H
Published: May 2006

In Drosophila modifying mutations of position-effect variegation have been successfully used to genetically dissect chromatin components. The enhancer of position-effect variegation E(var)3-93D [formerly E-var(3)3] encodes proteins containing a domain common to the transcriptional regulators tramtrack and the products of the Broad complex. It interacts with a number of chromatin genes that suppress position-effect variegation. Mutations in E(var)3-93D exhibit an imprinting-like effect on the Y chromosome. This effect is transmitted paternally over several generations. Homeotic transformations in E(var)3-93D mutants indicate an involvement of the gene products in regulation of homeotic gene complexes. An antiserum raised against E(var)3-93D protein detects this chromosomal protein in a large subset of sites in polytene chromosomes. Our genetic and molecular data suggest that the proteins of E(var)3-93D are generally involved in establishing and/or maintaining an open chromatin conformation.
Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases.
Authors: Edvardsen K , Chen W , Rucklidge G , Walsh F S , Obrink B , Bock E
Published: May 2006

During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix.
Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.
Authors: Burbelo P D , Utani A , Pan Z Q , Yamada Y
Published: May 2006

We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.
Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism.
Authors: Fernandes A P , Nelson K , Beverley S M
Published: May 2006

Molecular evolutionary relationships within the protozoan order Kinetoplastida were deduced from comparisons of the nuclear small and large subunit ribosomal RNA (rRNA) gene sequences. These studies show that relationships among the trypanosomatid protozoans differ from those previously proposed from studies of organismal characteristics or mitochondrial rRNAs. The genera Leishmania, Endotrypanum, Leptomonas, and Crithidia form a closely related group, which shows progressively more distant relationships to Phytomonas and Blastocrithidia, Trypanosoma cruzi, and lastly Trypanosoma brucei. The rooting of the trypanosomatid tree was accomplished by using Bodo caudatus (family Bodonidae) as an outgroup, a status confirmed by molecular comparisons with other eukaryotes. The nuclear rRNA tree agrees well with data obtained from comparisons of other nuclear genes. Differences with the proposed mitochondrial rRNA tree probably reflect the lack of a suitable outgroup for this tree, as the topologies are otherwise similar. Small subunit rRNA divergences within the trypanosomatids are large, approaching those among plants and animals, which underscores the evolutionary antiquity of the group. Analysis of the distribution of different parasitic life-styles of these species in conjunction with a probable timing of evolutionary divergences suggests that vertebrate parasitism arose multiple times in the trypanosomatids.
Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.
Authors: Deber C M , Khan A R , Li Z , Joensson C , Glibowicka M , Wang J
Published: May 2006

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.
A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.
Authors: Bruand C , Le Chatelier E , Ehrlich S D , Jannière L
Published: May 2006

Plasmid pAM beta 1 from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM beta 1 minimal replicon shares no homology with highly conserved ColE1-type replicons, which use PolI for initiation but do not encode a Rep, or with ColE2 and ColE3 replicons, which require PolI for replication and encode a Rep. We propose that pAM beta 1 and a number of other naturally occurring and closely related plasmids from a distinct plasmid class.
Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain.
Authors: Lanza P , Billetta R , Antonenko S , Zanetti M
Published: May 2006

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.
Mevalonate availability and cardiovascular functions.
Authors: Roullet J B , Xue H , Pappu A S , Roullet C , Holcomb S , McCarron D A
Published: May 2006

Data delineating the relationship between disorders of cholesterol metabolism and elevated blood pressure (BP) do not exist. We postulated that mevalonate, the metabolic precursor of endogenous cholesterol and the direct product of 3-hydroxy-3-methylglutaryl-CoA reductase, was a contributing factor for the maintenance of vascular tone and systemic BP. We conducted in vivo, ex vivo, and in vitro experiments in normotensive and hypertensive rats, where exogenous mevalonate and lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, were used, respectively, to increase or limit mevalonate availability. Mevalonate decreased BP in the whole animal without significant change in plasma cholesterol. Incubation of aortas with mevalonate attenuated their reactivity to norepinephrine and increased their response to endothelium-dependent and -independent relaxing factors. Lovastatin, in contrast, had the opposite effect in vivo and in vitro: it increased BP, enhanced vascular response to norepinephrine, and impaired endothelium-dependent and -independent relaxations. Neither agent modified cholesterol vascular content. Alteration of vascular reactivity was also observed in resistance vessels from animals pretreated with lovastatin. Our findings suggest that mevalonate availability is an unrecognized metabolic contributor to vascular tone and BP. They imply that (i) metabolites of the mevalonate pathway other than cholesterol could potentially control vascular functions and cardiovascular hemodynamics, (ii) elevated arterial pressure could be in part the consequence of primary disorders of this pathway, and (iii) pharmacological inhibition of mevalonate production as a means to lower plasma cholesterol may have an adverse impact on other cardiovascular risk factors, such as BP.
Activation of human primary visual cortex during visual recall: a magnetic resonance imaging study.
Authors: Le Bihan D , Turner R , Zeffiro T A , Cuénod C A , Jezzard P , Bonnerot V
Published: May 2006

The degree to which the process involved in visual perception and visual imagery share a common neuroanatomical substrate is unclear. Physiological evidence for localization of visual imagery early in the visual pathways would have important bearing on current theories of visual processing. A magnetic resonance imaging technique sensitive to regional changes in blood oxygenation was used to obtain functional activation maps in the human visual cortex. During recall of a visual stimulus, focal increases in signal related to changes in blood flow were detected in V1 and V2 cortex in five of seven subjects. These experiments show that the same areas of the early visual cortex that are excited by visual stimulation are also activated during mental representation of the same stimulus. Some of the processes used in topographically mapped cortical areas during visual perception may also be utilized during visual recall.
Acyclovir diphosphate dimyristoylglycerol: a phospholipid prodrug with activity against acyclovir-resistant herpes simplex virus.
Authors: Hostetler K Y , Parker S , Sridhar C N , Martin M J , Li J L , Stuhmiller L M , van Wijk G M , van den Bosch H , Gardner M F , Aldern K A
Published: May 2006

Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine, Zovirax) is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovir-resistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (IC50 = 0.25 microM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 microM). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3H]acyclovir diphosphate dimyristoylglycerol relative to acyclovir. Acyclovir monophosphate added to the culture medium (outside the cell) did not circumvent the acyclovir resistance of the TK-negative DM21 mutant, presumably due to its conversion to acyclovir by phosphatases. Acyclovir diphosphate diacylglycerol prodrugs may be useful in treating TK-deficient mutant and wild-type strains of HSV.
Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits.
Authors: Rommelaere H , Van Troys M , Gao Y , Melki R , Cowan N J , Vandekerckhove J , Ampe C
Published: May 2006

We have characterized the cytosolic chaperonin from both rabbit reticulocyte lysate and bovine testis. The heteromeric complex contains eight subunits. Partial amino acid sequence data reveal that one of these is t-complex polypeptide 1 (TCP-1), while the other seven are TCP-1-related polypeptides, implicating the existence of a multigene family of TCP-1 homologues. We provide evidence that TCP-1 ring complex from bovine testis can facilitate the folding of both actin and tubulin, although, as in the case of chaperonin from reticulocyte lysate, two cofactors are required for the generation of properly folded tubulin. An additional molecule of TCP-1 may associate with the chaperonin depending on the purification procedure used. We propose that a highly conserved region in these polypeptides and in other chaperonins of the cpn60 chaperone family participates in ATP binding.
Crystal structure of an oligonucleotide duplex containing G.G base pairs: influence of mispairing on DNA backbone conformation.
Authors: Skelly J V , Edwards K J , Jenkins T C , Neidle S
Published: May 2006

The structure of the G.G mispaired dodecanucleotide d(CGCGAATTGGCG)2 has been solved by x-ray crystallography and refined to an R factor of 18.8% at 2.2 A resolution for 3513 reflections. The dodecamer crystallizes as a B-type DNA double helix. It contains two G(anti).G(syn) base pairs--i.e., G-4/G-16(anti).G-21/G-9(syn). The Hoogsteen base pairing involves atoms O-6 and N-7 of the guanine in the syn conformation with atoms N-1 and N-2 of the anti-paired purine. One G.G base pair has a bifurcated hydrogen bond between G-4(N-1)...G-21(N-7) and G-4(N-1)...G-21(O-6). There is little overall structural distortion of the double helix induced as a consequence of the mispairing. The helical width is significantly increased by comparison with the structure of the native duplex, and the minor groove width in the 5'-AATT region is decreased. The G.G base pairing induces high-BII phosphate conformations at residues G-9 and T-20 in addition to more normal BII conformations at G-10 and G-22. It is suggested that these backbone aberrations provide signals for the facile repairability of G.G mispairs in DNA.
Exploring the energy landscape in proteins.
Authors: Straub J E , Thirumalai D
Published: May 2006

We present two methods to probe the energy landscape and motions of proteins in the context of molecular dynamics simulations of the helix-forming S-peptide of RNase A and the RNase A-3'-UMP enzyme-product complex. The first method uses the generalized ergodic measure to compute the rate of conformational space sampling. Using the dynamics of nonbonded forces as a means of probing the time scale for ergodicity to be obtained, we argue that even in a relatively short time (< 10 psec) several different conformational substrates are sampled. At longer times, barriers on the order of a few kcal/mol (1 cal = 4.184 J) are involved in the large-scale motion of proteins. We also present an approximate method for evaluating the distribution of barrier heights g(EB) using the instantaneous normal-mode spectra of a protein. For the S-peptide, we show that g(EB) is adequately represented by a Poisson distribution. By comparing with previous work on other systems, we suggest that the statistical characteristics of the energy landscape may be a "universal" feature of all proteins.
Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles.
Authors: Rymond B C
Published: May 2006

The PRP38 gene of Saccharomyces cerevisiae is necessary for the excision of intron sequences from pre-mRNA and required for the maintenance of maximal levels of U6 small nuclear RNA (snRNA). This report describes the identification of a gene of related function, SMD1, located immediately 3' to PRP38. The PRP38 and SMD1 transcription units are configured in an unusual "tail-to-tail" arrangement with their respective open reading frames terminating on opposite strands of a common 6-bp region. The predicted SMD1 polypeptide, Smd1p, is 40% identical to the D1 protein of human small nuclear ribonucleoprotein particles. Experimentally induced depletion of Smd1p blocks the first step of splicing and results in growth arrest. In addition, the levels of the trimethylguanosine-capped spliceosomal snRNAs, U1, U2, U4, and U5, but not the Prp38p-sensitive U6 snRNA, decrease in response to Smd1p depletion. The cap structures of snRNAs persisting in the absence of SMD1 expression appear to be peculiar, as they are poorly recognized by an anti-trimethylguanosine antibody. These data establish Smd1p as a required component of the cellular splicing apparatus and a factor in snRNA maturation and stability.
Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways.
Authors: Miao G H , Hong Z , Verma D P
Published: May 2006

We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library. The two sequences share 90% sequence homology in the coding regions. The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean. Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency. Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic. The relative expression level of the two soybean p34cdc2 genes varies in different tissues. Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways. Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis. This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection. Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones.
Design of a metallo-bZIP protein that discriminates between CRE and AP1 target sites: selection against AP1.
Authors: Cuenoud B , Schepartz A
Published: May 2006

The bZIP class of dimeric DNA binding proteins is characterized by a motif containing two discrete domains: a DNA contact domain defined by conserved basic and hydrophobic residues (basic domain), and a dimerization domain identified by a heptad repeat of leucine residues (zipper domain). Molecules are constructed in which the GCN4 dimerization domain is replaced by a series of stereochemically defined metal ion complexes that alter systematically the relative orientation of the basic domain peptides. Both the affinity and the specificity of DNA binding are modulated by seemingly small changes in metal complex stereochemistry. Although GCN4 binds CRE (ATGACGTCAT) and AP1 (ATGACTCAT) target sites with comparable affinity, one metallo-bZIP peptide ([G29Ts]2Fe) prefers the CRE by 4 kcal.mol-1 (1 cal = 4.184 J). Competition experiments performed with several DNAs demonstrate that discrimination between CRE and AP1 is dominated by selection against the AP1 site.
T-cell receptor V beta expression in normal human skin.
Authors: Dunn D A , Gadenne A S , Simha S , Lerner E A , Bigby M , Bleicher P A
Published: May 2006

The skin-associated immune system is the first line of defense against pathogenic attack from the environment and is simultaneously tolerant to localized autoantigens and to antigens of the normal microbial flora. The T-cell receptor (TCR) repertoire of skin lymphocytes may therefore be influenced by the skin microenvironment. We studied the expression of TCR beta-chain variable region (V beta) genes in normal skin by a polymerase chain reaction-based comparative method. When comparing the amplification of V beta genes in peripheral blood and normal skin, we found that TCR V beta 1, -7, -14, and -16 were often highly expressed in skin relative to peripheral blood, whereas V beta 5.1 was often highly expressed in peripheral blood mononuclear cells but not in skin. These results demonstrate that the TCR repertoire of skin lymphocytes is not determined by random sampling of peripheral blood mononuclear cells but may be molded by the interaction with self antigens and/or the normal microbial flora in the microenvironment of the skin.
Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis.
Authors: Tibayrenc M , Neubauer K , Barnabé C , Guerrini F , Skarecky D , Ayala F J
Published: May 2006

We have assayed genetic polymorphisms in several species of parasitic protozoa by means of random amplified polymorphic DNA (RAPD). One goal was to ascertain the suitability of RAPD markers for investigating genetic and evolutionary problems, particularly in organisms, such as the parasitic protozoa, unsuitable for traditional methods of genetic analysis. Another goal was to test certain hypotheses concerning Trypanosoma cruzi, and other protozoa, that have been established by multilocus enzyme electrophoresis. The RAPD results corroborate the hypothesis that the population structure of T. cruzi is clonal and yield a phylogeny of the clonal lineages in agreement with the one obtained by enzyme electrophoresis. This parity between the two sets of results confirms that RAPD markers are reliable genetic markers. The RAPD markers are also suitable for reconstructing species phylogenies and as diagnostic characters of species and subspecific lineages. The number of DNA polymorphisms that can be detected by the RAPD method seems virtually unlimited, since the number of primers can be increased effectively at will. The RAPD method is well suited for investigating genetic and evolutionary questions in certain organisms, because it is cost effective and demands no previous genetic knowledge about the organism.
Identification of a functional silencer element involved in neuron-specific expression of the synapsin I gene.
Authors: Li L , Suzuki T , Mori N , Greengard P
Published: May 2006

We have identified a functional silencer element (positions -231 to -211) in the human synapsin I gene that selectively represses its transcription in nonneuronal cells. Transfection experiments using synapsin I-luciferase constructs show that site-specific mutations or deletion of this silencer sequence results in expression of the reporter gene in nonneuronal cells. Moreover, the silencer element is capable of conferring repression on a heterologous promoter in nonneuronal cells. Gel-shift assays reveal the presence of a sequence-specific synapsin I silencer-binding protein in nonneuronal cell extracts but not in neuronal cell extracts. Mutagenesis studies of the silencer sequence demonstrate that formation of the specific silencer-protein complex in vitro correlates well with repression of transcription in vivo. These data indicate that the interaction between synapsin I silencer and its binding protein is involved in tissue-specific expression of the synapsin I gene. In addition, our results suggest the existence of at least one additional cis-acting element within the promoter-proximal region (positions -233 to +20) that also contributes to the neuron-specific expression of the synapsin I gene.
Identification of a nonprocessive telomerase activity from mouse cells.
Authors: Prowse K R , Avilion A A , Greider C W
Published: May 2006

Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.
Shared thematic elements in photochemical reaction centers.
Authors: Golbeck J H
Published: May 2006

The structural, functional, and evolutionary relationships between photosystem II and the purple nonsulfur bacterial reaction center have been recognized for several years. These can be classified as "quinone type" (type II) photosystems because the terminal electron acceptor is a mobile quinone molecule. The analogous relationship between photosystem I and the green sulfur bacterial (and helicobacterial) reaction centers has only recently become clear. These can be classified as "iron-sulfur type" (type I) photosystems because the terminal electron acceptor consists of one or more bound iron-sulfur clusters. At a fundamental level, the quinone type and iron-sulfur type reaction centers share a common photochemical motif in the early process of charge separation, leading to the speculation that all photochemical reaction centers have a common evolutionary origin. This review summarizes the current state of knowledge in comparative reaction center biochemistry between prokaryotic bacteria, cyanobacteria, and green plants.
Successful transient introduction of Leishmania RNA virus into a virally infected and an uninfected strain of Leishmania.
Authors: Armstrong T C , Keenan M C , Widmer G , Patterson J L
Published: May 2006

Viruses of Leishmania have recently been identified and characterized. These viruses are consistently double-stranded RNA viruses of approximately 5 kb. To date, they have not been reported to exist outside their protozoan host, nor have they been shown to be infectious. We report here the ability to transiently transfer these viruses to two strains of Leishmania, one previously infected and one that did not previously carry a virus. A PCR-based assay was used to detect viral negative-stranded RNA. Input RNA was ruled out as the source of template because a replication-incompetent (UV inactivated) virus was not detectable after transfer into Leishmania.
Molten-globule conformation of Arc repressor monomers determined by high-pressure 1H NMR spectroscopy.
Authors: Peng X , Jonas J , Silva J L
Published: May 2006

The conformation of the pressure-dissociated monomer of Arc repressor was characterized by 1H NMR spectroscopy. The NMR spectra of the monomer under pressure (up to 5.0 kbar; 1 bar = 100 kPa) are typical of a molten globule and they are considerably different from those of the native dimer and thermally denatured monomer. The two-dimensional nuclear Overhauser effect spectra suggest that the pressure-induced molten globule retains some secondary structure. The presence of nuclear Overhauser effects in the beta-sheet region in the dissociated state suggests that the intermonomer beta-sheet (residues 8-14) in the native dimer is replaced by an intramonomer beta-sheet. Changes in one-dimensional and two-dimensional NMR spectra prior to pressure dissociation were found and suggest the existence of a "predissociated" state.
Stromelysin 3 belongs to a subgroup of proteinases expressed in breast carcinoma fibroblastic cells and possibly implicated in tumor progression.
Authors: Wolf C , Rouyer N , Lutz Y , Adida C , Loriot M , Bellocq J P , Chambon P , Basset P
Published: May 2006

The expression of the stromelysin 3 (ST3) gene, which encodes a putative matrix metalloproteinase, was studied during breast cancer progression. The ST3 gene is expressed in all invasive breast carcinomas, in a number of their metastases, and in some in situ carcinomas where the probability of detecting ST3 transcripts correlates with the known risk of these carcinomas to become invasive. ST3 RNA and protein were specifically detected in fibroblastic cells immediately surrounding the neoplastic cells in both primary and metastatic tumors. This expression pattern distinguishes the ST3 gene from other matrix metalloproteinase genes, most notably from the 72-kDa type IV collagenase gene, which can be expressed in fibroblastic cells distributed throughout the stroma of primary breast carcinomas. Furthermore, high levels of 72-kDa type IV collagenase, but not of ST3 transcripts, are detected in benign breast fibroadenomas. Interestingly, the urokinase and ST3 genes exhibit very similar patterns of expression in breast carcinomas, which suggests that their products may cooperate during cancer progression.
The emergence of new DNA repeats and the divergence of primates.
Authors: Minghetti P P , Dugaiczyk A
Published: May 2006

We have identified four genetic novelties that are fixed in specific primate lineages and hence can serve as phylogenetic time markers. One Alu DNA repeat is present in the human lineage but is absent from the great apes. Another Alu DNA repeat is present in the gorilla lineage but is absent from the human, chimpanzee, and orangutan. A progenitor Xba1 element is present in the human, chimpanzee, gorilla, and orangutan, but only in the human lineage did it give rise to a transposed progeny, Xba2. The saltatory appearance of Xba2 is an example of a one-time event in the evolutionary history of a species. The enolase pseudogene, known to be present as a single copy in the human, was found to be present in four other primates, including the baboon, an Old World monkey. Using the accepted value of 5 x 10(-9) nucleotide substitutions per site per year as the evolutionary rate for pseudogenes, we calculated that the enolase pseudogene arose approximately 14 million years ago. The calculated age for this pseudogene and its presence in the baboon are incongruent with each other, since Old World monkeys are considered to have diverged from the hominid lineage some 30 million years ago. Thus the rate of evolution in the enolase pseudogene is only about 2.5 x 10(-9) substitutions per site per year, or half the rate in other pseudogenes. It is concluded that rates of substitution vary between species, even for similar DNA elements such as pseudogenes. We submit that new DNA repeats arise in the genomes of species in irreversible and punctuated events and hence can be used as molecular time markers to decipher phylogenies.
Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein.
Authors: Reines D , Mote J
Published: May 2006

In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments.
Orientation of the LexA DNA-binding motif on operator DNA as inferred from cysteine-mediated phenyl azide crosslinking.
Authors: Dumoulin P , Oertel-Buchheit P , Granger-Schnarr M , Schnarr M
Published: May 2006

To address the question how the recognition helix of the LexA repressor is positioned within the major groove of operator DNA we have applied a site-specific photocrosslinking approach using a LexA mutant repressor (LexA-C52) that harbors a single cysteine side chain in position 52, close to the COOH terminus of helix 3. The LexA-C52 mutant repressor has been purified and modified site-specifically with the photoreactive azido compound 4-azidophenacyl bromide, giving rise to LexA-C52*. Here we show that LexA-C52* may be selectively photocrosslinked with two adjacent bases within each operator half-site. The crosslinked bases are located, respectively, 10 and 11 base pairs from the dyad axis of the operator. The crosslinking data imply that the LexA recognition helix is oriented opposite to what is generally observed for helix-turn-helix proteins and that this helix should form a steeper angle with respect to the plane of the base pairs than is observed for standard helix-turn-helix proteins.
Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.
Authors: Johara M , Toyoshima Y Y , Ishijima A , Kojima H , Yanagida T , Sutoh K
Published: May 2006

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.
Brain contains two forms of synaptic vesicle protein 2.
Authors: Bajjalieh S M , Peterson K , Linial M , Scheller R H
Published: May 2006

Molecular cloning of a cDNA encoding synaptic vesicle protein 2 (SV2) revealed that it is homologous to a family of proton cotransporters from bacteria and fungi and to a related family of glucose transporters found in mammals. The similarity to proton cotransporters raised the possibility that SV2 might mediate the uptake of neurotransmitters into vesicles, an activity known to require a proton gradient. To determine whether SV2 is a member of a family of vesicular proteins, we used the SV2 clone to screen for similar cDNAs in rat brain. We characterized 42 clones, 25 of which encode SV2 and 4 of which encode a protein, SV2B, that is 65% identical and 78% similar to SV2. The protein encoded by SV2B cDNA is recognized by the monoclonal antibody that defines the SV2 protein. When SV2B is expressed in COS cells, antibody labeling is reticular in nature, suggesting that SV2B, like SV2 (hence, SV2A), is segregated to intracellular membranes. The expression of SV2B is limited to neural tissue. While both forms of SV2 are expressed in all brain regions, SV2B is expressed at highest levels in the cortex and hippocampus, whereas the highest level of expression of SV2A is in subcortical regions. Therefore, the SV2 proteins, like other characterized synaptic vesicle proteins, comprise a small gene family.
Activity of purified NIFA, a transcriptional activator of nitrogen fixation genes.
Authors: Lee H S , Berger D K , Kustu S
Published: May 2006

The NIFA protein activates transcription of nitrogen fixation (nif) operons by the sigma 54-holoenzyme form of RNA polymerase. We purified active NIFA from Klebsiella pneumoniae in the form of a maltose-binding protein (MBP)-NIFA fusion; proteolytic release of MBP yielded inactive and insoluble NIFA. MBP-NIFA activated transcription from the nifHDK promoter in a purified transcription system. Like the related transcriptional activator NTRC, MBP-NIFA catalyzed the ATP-dependent isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. MBP-NIFA had a broader nucleotide specificity than NTRC, being able to utilize pyrimidine in addition to purine nucleoside triphosphates. Both MBP-NIFA and a purified C-terminal fragment of NIFA bound to the upstream activation sequence for the nifHDK promoter, as assessed by DNAse I footprinting. When assays were performed at 37 degrees C instead of the usual 30 degrees C, transcriptional activation, open complex formation, and DNA binding by MBP-NIFA were all abolished, consistent with the known heat lability of NIFA. However, the purified C-terminal fragment of NIFA still bound the upstream activation sequence at 37 degrees C, indicating that the function of the helix-turn-helix DNA-binding motif is not inherently heat-labile.