bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence analysis of the complete cDNA and encoded polypeptide for the Glued gene of Drosophila melanogaster.
Authors: Swaroop A , Swaroop M , Garen A
Published: May 2006

The complete cDNA sequence for the Glued gene of wild-type Drosophila melanogaster contains an open reading frame encoding 1319 amino acids, which constitute the Glued polypeptide. The secondary predicted from the deduced sequence of the Glued polypeptide has extensive alpha-helical internal domains, which contain heptad-repeat sequences characteristic of an elongated coiled-coil conformation. There are striking sequence and conformation similarities between the Glued alpha-helical domains and those found in certain filamentous proteins from various organisms, particularly in muscle fibers and intermediate filaments. The possible role of the Glued polypeptide as an architectural filamentous component of Drosophila cells and tissues is discussed. Two of the five Glued exons are located in the 5' untranslated region of the cDNA. One of the introns interrupting the Glued open reading frame encodes at least two polyadenylylated transcripts, suggesting that other genes might map within the span of the Glued gene.
RNase III stimulates the translation of the cIII gene of bacteriophage lambda.
Authors: Altuvia S , Locker-Giladi H , Koby S , Ben-Nun O , Oppenheim A B
Published: May 2006

The bacteriophage lambda cIII gene product regulates the lysogenic pathway by stabilizing the lambda cII regulatory protein. Our results show that the expression of the lambda cIII gene is subject to specific requirements. Tests of a set of cIII-lacZ gene and operon fusions reveal that a sequence upstream of the cIII ribosome binding site is needed for cIII translation. The sequence contains an inefficient RNase III processing site. Furthermore, expression of cIII is drastically reduced in cells lacking RNase III. We have isolated a phage carrying a mutation (r1), which lies in the upstream sequence, that leads to a reduction in cIII translation and inactivates the RNase III processing site. The r1 mutant is nevertheless still dependent on RNase III for cIII translation; r1 reduces cIII translation by a factor of 3 in wild-type cells and by a factor of approximately equal to 30 in an RNase III mutant host. We propose that RNase III stimulates cIII translation by binding to the upstream sequence and thereby exposing the cIII ribosome binding site. This stimulation does not involve RNA cleavage. Consistent with this hypothesis is our finding that, in vitro, unprocessed cIII mRNA is translated, whereas RNase III-cleaved cIII mRNA is not.
Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes.
Authors: Smith M M , Merlie J P , Lawrence J C
Published: May 2006

By using 32P-labeling methods and performing immunoprecipitations with specific antibodies, we have found that three subunits of the nicotinic acetylcholine receptor are phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in alpha, beta, and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the beta subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the beta subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and beta subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca2+-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.
DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene
Authors: Walker John C. , Howard Elizabeth A. , Dennis Elizabeth S. , Peacock W. James
Published: May 2006

Expression of the maize alcohol dehydrogenase 1 (
Adh1
) gene is transcriptionally regulated under conditions of anaerobic stress. DNA sequences required for the expression of
Adh1
have been identified by a functional analysis of
in vitro
constructed hybrid genes consisting of the
Adh1
promoter fused to the chloramphenicol acetyltransferase coding region. A series of 5′ deletions, 3′ deletions, hybrid promoters, and linker scanning mutants of the
Adh-CAT
hybrid gene were introduced into maize protoplasts by electroporation and assayed for chloramphenicol acetyltransferase activity after incubation of the protoplasts under different oxygen tensions. The results indicate that a 40-base-pair DNA sequence within the
Adh1
promoter is required for anaerobically regulated expression of the hybrid gene. Clustered point mutations in this sequence show that it is composed of two essential regions, each ≈15 base pairs, separated by a 10-base-pair DNA sequence that does not appear to be important for anaerobic expression. Attachment of this 40-base-pair element to an unrelated promoter shows that this DNA sequence is both necessary and sufficient for induction of gene expression by low oxygen stress.
Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5).
Authors: Campbell H D , Tucker W Q , Hort Y , Martinson M E , Mayo G , Clutterbuck E J , Sanderson C J , Young I G
Published: May 2006

The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approximately 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.
Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.
Authors: Matthews B W , Nicholson H , Becktel W J
Published: May 2006

It is proposed that the stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa----Pro and the other of the form Gly----Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82----Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77----Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute approximately 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.
Molecular cloning and amino acid sequence of leukotriene A4 hydrolase.
Authors: Funk C D , Rådmark O , Fu J Y , Matsumoto T , Jörnvall H , Shimizu T , Samuelsson B
Published: May 2006

A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes.
Constitutive expression of a gene encoding a polypeptide homologous to biologically active human platelet protein in Rous sarcoma virus-transformed fibroblasts.
Authors: Bedard P A , Alcorta D , Simmons D L , Luk K C , Erikson R L
Published: May 2006

A molecular clone corresponding to a 1.2-kilobase mRNA enriched in Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF) was identified by differential screening of a cDNA library. The induction of the cloned sequence (denoted pCEF-4) in CEF infected by the temperature-sensitive mutant NY72-4 Rous sarcoma virus is rapid and independent of protein synthesis. DNA sequencing of the 1.2-kilobase insert of CEF-4 revealed an open reading frame that predicts an 11-kDa protein. The predicted pCEF-4 gene product is homologous to human connective tissue-activating peptide III (CTAP-III) and platelet factor 4 (PF-4). Serum stimulation of quiescent normal CEF results in a rapid but transient expression of pCEF-4 mRNA. Hence, pCEF-4 mRNA is expressed at the G0-G1 transition and during the first G1 phase of normal CEF reentering the cell cycle. The expression of pCEF-4 mRNA in Rous sarcoma virus-transformed CEF appears to be the result of transcriptional activation and stabilization of the transcript.
Expression of a set of synthetic suppressor tRNA(Phe) genes in Saccharomyces cerevisiae.
Authors: Masson J M , Meuris P , Grunstein M , Abelson J , Miller J H
Published: May 2006

Synthetic ochre and amber tRNA suppressor genes derived from the yeast tRNA(PheGAA) sequence have been constructed. They were efficiently transcribed in vitro and expressed in vivo via a synthetic expression cassette. tRNA(PheUUA) and tRNA(PheUUA) delta IVS (IVS = intervening sequence) are relatively inefficient ochre suppressors. They are toxic to the cell when expressed on a multicopy plasmid, and they do not suppress at all when present as single copies. The intron does not seem to have any effect on suppression. In contrast, the amber suppressor tRNA(PheCUA) delta IVS is efficient when expressed from a single-copy plasmid, while its efficiency is reduced on a multicopy vector.
Heterologous enzyme function in Escherichia coli and the selection of genes encoding the catalytic RNA subunit of RNase P.
Authors: Lawrence N P , Richman A , Amini R , Altman S
Published: May 2006

The gene for the catalytic RNA subunit of RNase P has been isolated from several Enterobacteriaceae by complementation of an Escherichia coli strain that is temperature-sensitive for RNase P activity. The selection procedure relies on the ability of the heterologous gene products to function enzymatically in E. coli. This procedure obviates the need for positive results in DNA blot hybridization experiments or for the purification of holoenzyme to identify the RNA component of RNase P and its corresponding gene from organisms other than E. coli. Comparisons of the variations in sequences provide the basis for a refined two-dimensional model of the secondary structure of M1 RNA.
Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop.
Authors: Seong B L , RajBhandary U L
Published: May 2006

We have generated mutants of Escherichia coli formylmethionine initiator tRNA in which one, two, and all three G X C base pairs in the GGGCCC sequence in the anticodon stem are changed to those found in E. coli elongator methionine tRNA. Overproduction of the mutant tRNAs using M13 recombinants as an expression vector and development of a one-step purification scheme allowed us to purify, characterize, and analyze the function of the mutant tRNAs. After aminoacylation and formylation, the function of mutant formylmethionyl tRNAs was analyzed in an MS2 RNA-directed in vitro protein-synthesizing system, in AUG-dependent ribosomal P site binding, and in initiation factor binding. The mutant tRNAs show progressive loss of activity in initiation, the mutant with all three G X C base pairs substituted being the least active. The mutations affect binding to the ribosomal P site. None of the mutations affects binding to initiation factor 2. We also show that there is a progressive increase in accessibility of phosphodiester bonds in the anticodon loop of the three mutants to S1 nuclease, such that the cleavage pattern of the mutant with all three G X C base-pair changes resembles that of elongator tRNAs. These results are consistent with the notion that the contiguous G X C base pairs in the anticodon stem of initiator tRNAs impart on the anticodon loop a unique conformation, which may be important in targeting the initiator tRNA to the ribosomal P site during initiation of protein synthesis.
Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane.
Authors: Murthy M S , Pande S V
Published: May 2006

The overt carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity of intact mitochondria from rat heart and liver was found to be resistant to the action of proteases such as Nagarse (subtilisin, EC 3.4.21.14). Nagarse under the same conditions, however, greatly decreased the malonyl-CoA inhibition of carnitine palmitoyltransferase activity, the high-affinity binding of malonyl-CoA to mitochondria, and the ability of malonyl-CoA to shift to the right the sigmoid activity curve of carnitine palmitoyltransferase observed with variations in palmitoyl-CoA concentration. No noticeable effect of Nagarse pretreatment was observed on the binding of octanoyl-CoA to mitochondria. Subfractionation of liver mitochondria using a combination of swelling, shrinking, and density gradient centrifugation yielded a membrane fraction in which the specific activities of the outer membrane marker enzymes were enriched greater than or equal to 16-fold together with a near-parallel enrichment of malonyl-CoA-inhibitable carnitine palmitoyltransferase activity. The percent recovery of this carnitine palmitoyltransferase in the outer membrane vesicles also matched that of the known outer membrane markers. The carnitine palmitoyltransferase activity of these out-side-out vesicles became susceptible to added Nagarse only on their cosonication. These findings show that whereas the malonyl-CoA binding site relevant to the inhibition of carnitine palmitoyltransferase is situated on the outer side of the outer membrane, the overt carnitine palmitoyltransferase activity resides on the inner side of the outer membrane.
A heat shock-induced, polymerase III-transcribed RNA selectively associates with polysomal ribosomes in Tetrahymena thermophila.
Authors: Kraus K W , Good P J , Hallberg R L
Published: May 2006

Tetrahymena thermophila cells, subjected to a heat shock-inducing temperature, manifest a number of translationally regulated changes during the course of a continuous heat shock treatment. One particular change, the resumption of translation of mRNAs coding for normal cellular proteins, was found to correlate with a polysomal ribosome association not found prior to heat shock. A low molecular weight RNA (ca. 270 nucleotides), whose rapid accumulation was induced by heat shock, became quantitatively associated with polysomal ribosomes during that time when normal cell protein synthesis became reestablished. We estimated that there were one or two of these RNAs per ribosome uniformly distributed throughout the polysomal ribosome population. The gene (or genes) coding for this RNA were found to be transcribed by polymerase III.
Cytochrome P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues.
Authors: Chung B C , Picado-Leonard J , Haniu M , Bienkowski M , Hall P F , Shively J E , Miller W L
Published: May 2006

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.
The primary structure of the myosin head.
Authors: Maita T , Hayashida M , Tanioka Y , Komine Y , Matsuda G
Published: May 2006

The sequence of the NH2-terminal 808 amino acid residues of chicken pectoralis muscle myosin head was determined. Three characteristic 20-, 23-, and 50-kDa fragments were isolated from a digest of myosin subfragment 1 (S1) by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine hydrochloride, followed by anion-exchange chromatography on a QAE-Sephadex A-50 column in the presence of 8 M urea. The fragments were sequenced completely by conventional methods. Peptides overlapping the 23- and 50-kDa fragments and also overlapping the 50- and 20-kDa fragments were obtained by cleaving S1 with cyanogen bromide. Comparison of the 23-kDa and 50-kDa sequences with that of the overlapping peptide indicated that no additional amino acid exists between the 23- and 50-kDa fragments and that 5 amino acids exist between the 50- and 20-kDa fragments of S1. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 35, epsilon-N-trimethyllysine residues at 130 and 550, and 3-N-methylhistidine at 754.
Supercoiling of the DNA template during transcription.
Authors: Liu L F , Wang J C
Published: May 2006

Transcription of a right-handed double-helical DNA requires a relative rotation of the RNA polymerase and its nascent RNA around the DNA. We describe conditions under which the resistance to the rotational motion of the transcription ensemble around the DNA can be large. In such cases, the advancing polymerase generates positive supercoils in the DNA template ahead of it and negative supercoils behind it. Mutual annihilation of the positively and negatively supercoiled regions may be prevented by anchoring points on the DNA to a large structure, or, in the case of an unanchored plasmid, by the presence of two oppositely oriented transcription units. In prokaryotes, DNA topoisomerase I preferentially removes negative supercoils and DNA gyrase (topoisomerase II) removes positive ones. Our model thus provides an explanation for the experimentally observed high degree of negative or positive supercoiling of intracellular pBR322 DNA when DNA topoisomerase I or gyrase is respectively inhibited. We discuss the implications of our model in terms of supercoiling regulation, DNA conformational transitions, and gene regulation in both prokaryotes and eukaryotes.
Infrared spectroscopic evidence of conformational transitions of an atrial natriuretic peptide.
Authors: Surewicz W K , Mantsch H H , Stahl G L , Epand R M
Published: May 2006

The conformational properties of the atrial natriuretic peptide atriopeptin III were investigated by Fourier-transform infrared spectroscopy. Infrared spectra in the amide I region were analyzed quantitatively using deconvolution and band-fitting procedures. According to this analysis, in aqueous solution the monomeric peptide has a random structure. Binding to bilayer vesicles of dimyristoyl phosphatidylglycerol results in drastic conformational changes. The lipid-complexed atriopeptin III adopts a highly ordered structure of predominantly beta-sheets. A transition to a similar, but not identical, beta-structure occurs upon self-association of the peptide. The results of model experiments suggest that the binding of this atrial peptide to the target cell membrane is associated with the induction of beta-sheet structure and that it is this latter conformation that is predominant in the active form of the hormone.
Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.
Authors: Smith F R , Gingrich D , Hoffman B M , Ackers G K
Published: May 2006

In a previous study on cyanomethemoglobin the 10 tetrameric species (each with a unique combination of ligated and unligated subunits) were found to exhibit three distinct free energies of cooperative interaction. The distribution of these free energies among the partially ligated species is incompatible with a two-state mechanism of molecular switching and requires a minimum of three molecular structures with distinctly different free energies of heme-heme interaction. Ligand-linked transitions between the three cooperativity states were found to be "combinatorial"--i.e., dependent upon changes in both the number and specific configuration of bound ligands. Here we present results from two other chemical systems that mimic intermediate oxygenation states. In these systems the heme iron is replaced by manganese in certain of the subunits. We find the same distribution of cooperative free energies as reported for the cyanomethemoglobin system. These results demonstrate that the three-state combinatorial nature of cooperative switching is neither a special feature of the cyanomet reactions nor of the substitution of manganese for iron, but reflects a fundamental property of hemoglobin. These findings are compared with crystallographic structural results on partially ligated hemoglobins.
Products of Leishmania braziliensis glucose catabolism: release of D-lactate and, under anaerobic conditions, glycerol.
Authors: Darling T N , Davis D G , London R E , Blum J J
Published: May 2006

Leishmania braziliensis panamensis promastigotes were incubated with glucose as the sole carbon source. About one-fifth of the glucose consumed under aerobic conditions was oxidized to CO2. Nuclear magnetic resonance studies with [1-13C]glucose showed that the other products released were succinate, acetate, alanine, pyruvate, and lactate. Under anaerobic conditions, lactate output increased, glycerol became a major product, and, surprisingly, glucose consumption decreased. Enzymatic assays showed that the lactate formed was D(-)-lactate. The release of alanine during incubation with glucose as the sole carbon source suggested that appreciable proteolysis occurred, consistent with our observation that a large amount of ammonia was released under these conditions. The discoveries that D-lactate is a product of L. braziliensis glucose catabolism, that glycerol is produced under anaerobic conditions, and that the cells exhibit a "reverse" Pasteur effect open the way for detailed studies of the pathways of glucose metabolism and their regulation in this organism.
Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry.
Authors: Assaraf Y G , Schimke R T
Published: May 2006

We have studied the frequency of transport mutations in methotrexate-resistant Chinese hamster ovary cells using a rapid-flow cytometric technique. After saturating cells with fluoresceinated methotrexate, we examined the ability of hydrophilic and lipophilic antifolates to displace fluoresceinated methotrexate binding to dihydrofolate reductase. Cells with methotrexate transport deficiency are unable to take up methotrexate and thus retain the fluorescence, whereas the lipophilic antifolates displace fluoresceinated methotrexate equally well in sensitive and resistant cell lines. These resistant clones fail to take up methotrexate and occur with high frequencies upon single-step selections at methotrexate concentrations approximately equal to 7-fold the 50% killing concentration. The majority of such first-step resistant clones appear to derive their resistance solely from transport deficiency; they exhibit no overproduction of dihydrofolate reductase and no increase in either steady-state mRNA levels or gene copy number. Possible applications of the use of fluoresceinated methotrexate to the characterization of various mechanisms of methotrexate resistance in mixed cell populations are discussed.
Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number.
Authors: Tschumper G , Carbon J
Published: May 2006

Two yeast (Saccharomyces cerevisiae) mutants that tolerate centromere (CEN) plasmids at high copy number have been isolated. The mutations relieve the restraint normally imposed on plasmid copy number by a cloned CEN sequence. Our CEN plasmids specify resistance to G418 and are high copy plasmids only when the mutant host cells are grown on medium containing this antibiotic. The high copy number of the plasmids is independent of the specific cloned CEN sequence and recovered plasmids show no alteration in structure or function of the CEN DNA. The efficiency with which CEN plasmids go to high copy number is increased if the mutant cell is cotransformed by another CEN plasmid. The genomic mutation responsible for the high copy number (COP) is dominant and stable, and it segregates in a Mendelian manner. Homozygous COP/COP a/alpha diploids do not tolerate CEN plasmids at high copy number, suggesting that the mutation is regulated by mating type. The genomic DNA from both mutant cells contains an altered transposon (Ty) restriction fragment that cosegregates with the COP phenotype in crosses of mutant and wild-type strains. The mutations may be transposon-mediated events that identify a gene involved in centromere or mitotic spindle function.
Tumor necrosis factor amplifies measles virus-mediated Ia induction on astrocytes.
Authors: Massa P T , Schimpl A , Wecker E , ter Meulen V
Published: May 2006

We describe the induction of Ia on cultured astrocytes by measles virus and the amplification of this induction by tumor necrosis factor (TNF). Measles virus induces Ia on rat astrocytes by direct interaction with these cells. TNF does not induce significant levels of Ia at any dose from 1 to 10,000 units/ml. As little as 10 units of TNF per ml, however, amplifies Ia-inducing signals generated by measles virus in astrocytes. In contrast, TNF and measles virus induce class I major histocompatibility complex (MHC) antigens, when applied individually, and TNF amplification of measles virus class I MHC induction is not apparent. The induction of either Ia or class I MHC antigens on rat astrocytes by measles virus does not depend on glial-derived soluble factors generated during infection. Since brain cells are normally lacking MHC antigens upon which T cells depend for interaction with antigen presenting cells, these data indicate that the ability of measles virus to directly stimulate MHC antigen expression and the ability of TNF to amplify Ia expression locally in the brain may be important in initiating cell-mediated immune response to viral infection.
Reduced expression of multiple forms of the alpha subunit of the stimulatory GTP-binding protein in pseudohypoparathyroidism type Ia.
Authors: Carter A , Bardin C , Collins R , Simons C , Bray P , Spiegel A
Published: May 2006

We examined the expression of the alpha subunit of the stimulatory GTP-binding protein (Gs) in fibroblasts of subjects with pseudohypoparathyroidism (PHP) type Ia by transfer blot hybridization and S1 nuclease analyses. Six subjects with PHP type Ia showed decreased steady-state content of Gs alpha mRNA. S1 nuclease analysis indicates that both long and short forms of Gs alpha mRNA are decreased, with no apparent change in the ratio of long to short forms in PHP compared with normal individuals. It appears likely that in some cases of PHP type Ia the genetic lesion affects the maintenance of mRNA levels for all forms of the Gs alpha subunit.
Deficits in human visual spatial attention following thalamic lesions.
Authors: Rafal R D , Posner M I
Published: May 2006

There has been speculation concerning the role that thalamic nuclei play in directing attention to locations in visual space [Crick, F. (1984) Proc. Natl. Acad. Sci. USA 81, 4586-4590]. We measured covert shifts of visual attention in three patients with unilateral thalamic hemorrhages shortly after the lesion and after a 6-month recovery period. The experiment measured reaction time to targets that occurred at locations to which attention had been cued (valid trials) or at a currently unattended location (invalid trials). Although the patients showed no deficits in visual fields with perimetry and no neglect in the 6-month follow-up, we found slow reaction times for targets on the side contralateral to the lesion whether or not attention had been cued to that location. Deficits have also been found in this task with cortical and midbrain lesions, but the patterns of performance are quite different. The results with thalamic patients suggest they have a specific deficit in the ability to use attention to improve the efficiency of processing visual targets contralateral to the lesion (engage operation). This finding is in accord with hypotheses of a thalamic link between cortical visual attention and pattern recognition systems proposed by Crick.
Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.
Authors: Prchal J T , Morley B J , Yoon S H , Coetzer T L , Palek J , Conboy J G , Kan Y W
Published: May 2006

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.
Spin glasses and the statistical mechanics of protein folding.
Authors: Bryngelson J D , Wolynes P G
Published: May 2006

The theory of spin glasses was used to study a simple model of protein folding. The phase diagram of the model was calculated, and the results of dynamics calculations are briefly reported. The relation of these results to folding experiments, the relation of these hypotheses to previous protein folding theories, and the implication of these hypotheses for protein folding prediction schemes are discussed.
Lysinuric protein intolerance mutation is expressed in the plasma membrane of cultured skin fibroblasts.
Authors: Smith D W , Scriver C R , Tenenhouse H S , Simell O
Published: May 2006

Lysinuric protein intolerance (LPI) is an autosomal recessive phenotype consistent with impaired transport of cationic amino acids at the basolateral membrane of intestinal and renal epithelia. On the assumption that the basolateral membrane of epithelial cells and plasma membrane of parenchymal cells are functional analogues, we studied transport of cationic amino acids by cultured skin fibroblasts from LPI and control subjects matched for age, sex, and site of biopsy. We measured Na+-independent transport of radiolabeled lysine, arginine, ornithine, and homoarginine on system y+, the carrier with preference for cationic amino acids, and leucine transport on system L (as the internal control). LPI cells had increased net uptake of cationic amino acids (nmol/mg of protein) relative to leucine. LPI cells also maintained increased steady-state intracellular pools of cationic amino acids. Neither increased metabolic utilization nor increased pool size were responsible for high uptake of cationic amino acids in LPI cells. We then measured trans-stimulated efflux of homoarginine as a specific test of system y+ activity. Homoarginine efflux was significantly impaired in LPI cells (P less than 0.05), whereas leucine efflux was similar in LPI and control cells. Percent trans-stimulation of homoarginine efflux was 1.0 +/- 0.5% in homozygous LPI cells, 10 +/- 0.5% in heterozygous cells, and 22 +/- 0.5% in control cells indicating a gene-dosage effect. The LPI mutation affects system y+ asymmetrically, selectively impairing efflux in fibroblast plasma membrane. To our knowledge, this appears to be the first demonstration that the skin fibroblast can be used to study a corresponding transport defect in intestinal and renal membranes.
Release of autocrine growth factor by primary and immortalized Schwann cells.
Authors: Porter S , Glaser L , Bunge R P
Published: May 2006

Schwann cells derived from neonatal rat sciatic nerve are quiescent in culture unless treated with specific mitogens. The use of glial growth factor (GGF) and forskolin has been found to be an effective method for stimulating proliferation of Schwann cells on a poly(L-lysine) substratum while maintaining their ability to myelinate axons in vitro. We find that repetitive passaging of Schwann cells with GGF and forskolin results in the loss of normal growth control; the cells are able to proliferate without added mitogens. The immortalized cells grow continuously in the absence of added growth factor and in the presence or absence of serum yet continue to express distinctive Schwann cell-surface antigens. The cells can associate with axons in culture, deposit a basal lamina, and ensheath axons, but they gradually lose their capacity to myelinate axons. The immortalized cells release growth-promoting activity into their culture medium. The released activity is effective in stimulating proliferation of primary Schwann cells that retain normal growth properties. Extracellular matrix molecules (laminin and fibronectin) augment the response of primary Schwann cells to the secreted mitogen. Quiescent primary Schwann cells also secrete a growth factor into their culture medium, but its activity is detectable only in the presence of added laminin or fibronectin. The results suggest that both normal and immortalized Schwann cells secrete an autocrine growth factor. Response to the autocrine factor appears to entail a multicomponent mechanism. Unlike primary cells, immortalized Schwann cells have the capacity to secrete all of the necessary components and to respond to them constitutively.
Capsid protein VP4 of poliovirus is N-myristoylated.
Authors: Paul A V , Schultz A , Pincus S E , Oroszlan S , Wimmer E
Published: May 2006

Poliovirus was labeled in vivo with [3H]myristic acid. Analysis of the capsid polypeptides revealed that the [3H]myristic acid residues copurified with VP4, the smallest and internal capsid protein of the virion. Evidence is presented showing unambiguously that the N-terminal glycine residue of VP4 is N-myristoylated. A previous analysis of the tryptic peptides of VP4 [Dorner, A. J., Dorner, L. F., Larsen, G. R., Wimmer, E. & Anderson, C. W. (1982) J. Virol. 42, 1017-1028] had shown that the N-terminal blocking group exists on all VP4 molecules as well as on VP0 and P1, two precursor polypeptides to VP4 in poliovirus. The possible function of the myristic acid residue in VP4 and in its precursor in poliovirus proliferation is discussed.
Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding.
Authors: Böni-Schnetzler M , Pilch P F
Published: May 2006

Epidermal growth factor (EGF) receptor monomers and noncovalently associated dimers were isolated by sucrose density gradient centrifugation, and their respective binding and autophosphorylation activities were determined. We find that monomers are low-affinity receptors and dimers are high-affinity receptors. In the absence of EGF, dimers exhibit a 4-fold higher autophosphorylation activity than do monomers. Addition of EGF increases autophosphorylation on monomers an average of 4.8-fold but has a minimal effect on autophosphorylation of dimers. Furthermore, EGF binding shifts the receptor monomer-dimer equilibrium to the dimer form. We conclude that EGF stimulates in vitro receptor autophosphorylation by inducing kinase-inactive receptor monomers to associate and form receptor dimers, in which conformation the autophosphorylation activity is enhanced.
Amino acid substitutions in inherited albumin variants from Amerindian and Japanese populations.
Authors: Takahashi N , Takahashi Y , Isobe T , Putnam F W , Fujita M , Satoh C , Neel J V
Published: May 2006

We report an effort to determine the basis for the altered migration of seven inherited albumin variants detected by one-dimensional electrophoresis in population surveys involving tribal Amerindians and Japanese children. An amino acid substitution has thus far been determined for four of the variants. The randomness in the albumin polypeptide of these and the other sixteen independently ascertained amino acid substitutions of albumin and proalbumin thus far established was analyzed; the clustering of eight of these at two positions in the six-amino acid propeptide sequence seems noteworthy. By comparison with other proteins studied by electrophoresis, albumin exhibits "average" variability. It is a paradox that individuals who, for genetic reasons, lack albumin exhibit no obvious ill effects; yet, electrophoretic variants of albumin are no more numerous than are variants of proteins, the absence of which results in severe disease.
Structural alterations of DNA ligase I in Bloom syndrome.
Authors: Willis A E , Weksberg R , Tomlinson S , Lindahl T
Published: May 2006

Cell lines derived from seven patients with Bloom syndrome all contain a DNA ligase I with unusual properties. Six lines were shown to have a reduced level of this enzyme activity and the residual enzyme was anomalously heat-labile. The seventh line contained a dimeric rather than monomeric form of ligase I. Several cell lines representative of other inherited human syndromes have apparently normal DNA ligases. The data indicate that Bloom syndrome is due to a defect in the structure of DNA ligase I caused by a "leaky" point mutation occurring at one of at least two alternative sites.
Sexual behavior: its genetic control during development and adulthood in Drosophila melanogaster.
Authors: Belote J M , Baker B S
Published: May 2006

Courtship behavior in Drosophila melanogaster males is an innate behavior pattern. Whether or not a fly will display male courtship behavior is governed by the action of a set of regulatory genes that control all aspects of somatic sexual differentiation. The wild-type function of one of these regulatory genes, transformer-2 (tra-2), is necessary for female sexual differentiation; in the absence of tra-2+ function XX individuals differentiate as males. A temperature-sensitive tra-2 allele has been used to investigate, by means of temperature shifts, when and how male courtship behavior is specified during development. The removal of tra-2ts function in the adult (by a shift of the tra-2ts mutant flies to the restrictive temperature) can lead to the appearance of male courtship behavior in flies that otherwise would not display these behaviors. These experiments suggest that the regulatory hierarchy controlling sexual differentiation is functioning in the adult central nervous system. More importantly, these results suggest that the adult central nervous system has some functional plasticity with respect to the innate behavioral pattern of male courtship and is maintained in a particular state of differentiation by the active control of gene expression in the adult.
Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene.
Authors: Atcheson C L , DiDomenico B , Frackman S , Esposito R E , Elder R T
Published: May 2006

The SPO11 gene, required for meiotic recombination in Saccharomyces cerevisiae, has been cloned by direct selection for complementation of the spo11-1 phenotype: lack of meiotic recombination and low spore viability. DNA sequencing indicates that the gene encodes a 398-amino acid protein having a predicted molecular mass of 45.3 kDa. There is no significant similarity between the SPO11 protein and other protein sequences, including those from genes known to be involved in DNA recombination or repair. Strains bearing a disruption allele are viable, indicating that SPO11 is dispensable for mitotic growth. RNA analyses demonstrate that SPO11 produces a 1.5-kilobase transcript that is developmentally regulated and expressed early in the sporulation process.
Cyclic AMP regulates the proportion of functional acetylcholine receptors on chicken ciliary ganglion neurons.
Authors: Margiotta J F , Berg D K , Dionne V E
Published: May 2006

Previous studies have shown that the number of functional acetylcholine receptors (AcChoRs) on chicken ciliary ganglion neurons in culture is considerably smaller than the total number of AcChoRs detected on the neurons by labeled receptor probes. Here we use patch-clamp recording to show that a cAMP-dependent process enhances the AcCho response of the neurons by a mechanism likely to involve an increase in the number of functional AcChoRs. The increase occurs without requiring protein synthesis and without involving a detectable increase in the total number of AcChoRs on the cell surface measured with a labeled receptor probe. The results imply that the neurons have functional and nonfunctional pools of AcChoRs and that functional receptors can be recruited from intracellular receptors or from nonfunctional receptors on the cell surface by a cAMP-dependent process. A cAMP-dependent regulation of the number of functional neurotransmitter receptors would provide a reversible mechanism by which cell-cell interactions could modulate synaptic transmission in the nervous system.
Major role for neuronal death during brain development: refinement of topographical connections.
Authors: Catsicas S , Thanos S , Clarke P G
Published: May 2006

The precision of the topographic projection from the isthmo-optic nucleus (ION) to the retina has been examined in chicken embryos and chicks by the retrograde transport of a fluorescent carbocyanine dye from restricted retinal sites. At all ages, the labeled neurons are most numerous in the topographically appropriate part of the ION, but in younger embryos up to 49% of them are found outside this region. The distribution of these "aberrantly" projecting neurons is variable, but they generally occur throughout the entire ION. They all die during the ION's period of neuronal death, accounting for most of the 60% cell loss that then occurs. We therefore suggest that a major role of neuronal death during brain development is to reduce the imprecision of neuronal connections.
Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.
Authors: Touchot N , Chardin P , Tavitian A
Published: May 2006

Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rab1, -2, -3, and -4, encoding proteins homologous to the yeast YPT protein. Amino acid homology scores with YPT range from 75% for rab1 to 37% for rab4, whereas the homologies with p21 ras and other ras-related proteins are approximately equal to 30%, and homologous residues were clustered in the regions involved in GTP/GDP binding. Another striking similarity shared by the rab and the other ras-related proteins is the conservation of at least one cysteine residue near the carboxyl-terminal end involved in the membrane binding of the ras proteins. rab1 is a mammalian homolog of the yeast YPT gene, and the four rab genes constitute an additional branch of the ras gene superfamily that to our knowledge has not been described in higher eukaryotes.