bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis.
Authors: Snyder M , Elledge S , Davis R W
Published: May 2006

A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented.
Expression of the oncogene of avian reticuloendotheliosis virus in Escherichia coli and identification of the transforming protein in reticuloendotheliosis virus T-transformed cells.
Authors: Herzog N K , Bose H R
Published: May 2006

The genome of reticuloendotheliosis virus T (REV-T) includes a unique oncogene v-rel, which is transcribed in low amounts into a 3.0-kilobase subgenomic mRNA in REV-T-transformed lymphoid cells. To identify the v-rel protein, REV-T DNA sequences were cloned into bacterial plasmid vectors designed to achieve expression of foreign DNA sequences in Escherichia coli. Portions of the v-rel gene were joined to the 5' segment of the trpE gene. Upon induction of trpE with indoleacrylic acid, large amounts of trpE-v-rel fusion proteins were produced by the bacteria carrying these recombinant plasmids. Two trpE-v-rel fusion proteins were synthesized in E. coli, which collectively represent three-quarters of the predicted v-rel protein. Polyclonal antisera were generated to trpE-v-red fusion proteins. These antisera were used in immunoblotting experiments to identify a 57-kDa v-rel protein in REV-T-transformed lymphoid cells lines and REV-T-infected chicken embryo fibroblast cultures. The v-rel gene expressed in E. coli under lac control was found to produce a 56-kDa protein. Although REV-T-transformed and Marek disease virus-transformed lymphoid cells contain c-rel mRNA transcripts, a c-rel protein could not be detected with antisera directed against v-rel fusion proteins.
Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain.
Authors: Ferrero P , Santi M R , Conti-Tronconi B , Costa E , Guidotti A
Published: May 2006

An endogenous brain neuropeptide with 104 amino acid residues that modulates gamma-aminobutyric acid receptor function was termed DBI because it displaces diazepam from its specific brain binding sites. Tryptic digestion of DBI generates an octadecaneuropeptide (ODN) that is more potent than the parent compound in the displacement of specifically bound beta-[3H]carboline-3-carboxylate methyl ester [( 3H]BCCM) and in proconflict action (Vogel test in thirsty rats). The proconflict action of ODN is antagonized by the imidobenzodiazepinone Ro 15-1788, which is a specific antagonist of beta-carboline and benzodiazepine recognition sites. The ODN amino acid sequence is Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. The pharmacological properties associated with this sequence were confirmed by comparing the activity of ODN generated from tryptic digestion of DBI with that of ODN obtained by synthesis. Amidation of the terminal lysine of ODN produces a peptide (ODN-NH2) devoid of pharmacological activity. Three peptides containing the COOH-terminal segment of ODN were synthesized. All these peptides [Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys (octapeptide), Pro-Gly-Leu-Leu-Asp-Leu-Lys (heptapeptide), and Gly-Leu-Leu-Asp-Leu-Lys (hexapeptide)] express the displacing and proconflict actions of ODN. In primary cultures of cerebellar granule cells of rat, DBI, ODN, octapeptide, heptapeptide, and hexapeptide preferentially displace [3H]BCCM over [3H]flunitrazepam; moreover, they displace bound [3H]BCCM completely but [3H]flunitrazepam only by 50%. These data suggest that ODN includes a specific ligand for the gamma-aminobutyric acid receptor regulatory site occupied by beta-carbolines. Using rabbit antibodies directed against the NH2-terminal portion of ODN, we detected ODN-like material in rat brain homogenates. However, whether this material is identical to the ODN generated by tryptic digestion of DBI remains to be established.
Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda phage shuttle vector.
Authors: Glazer P M , Sarkar S N , Summers W C
Published: May 2006

In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a lambda phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient (5 phage per microgram of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12J/m2), 78,510 phage were rescued, of which 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C----T transition.
Decapeptides as effective agonists from L-amino acids biologically equivalent to the luteinizing hormone-releasing hormone.
Authors: Folkers K , Bowers C Y , Tang P F , Kubota M
Published: May 2006

Apparently, no agonist has been found that is comparable in potency to the luteinizing hormone-releasing hormone (LHRH) for release of LH and follicle-stimulating hormone (FSH) without substitutions with unnatural or D forms of natural amino acids. Of 139 known "agonist analogs" of LHRH, two were active in the range of 65%. The four LHRHs known to occur in nature involve a total of six amino acids (Tyr, His, Leu, Trp, Arg, Gln) in positions 5, 7, and 8. There are 16 possible peptides with these six amino acids in positions 5, 7, and 8, of which 4 are the known LHRHs, and 2 more were synthesized. We have synthesized the 10 new peptides and assayed 11 in vivo and in vitro, and we found not only 1 but a total of 5 that have activity equivalent to or greater than that of LHRH for the release of LH and/or FSH under at least one assay condition. These five are as follows: [His5,Trp7,Gln8]LHRH; [His5,Trp7,Leu8]LHRH; [His5,Trp7]LHRH; [Trp7]LHRH; [His5]LHRH. Two of these five agonists variably released relatively more FSH than LH. One or more of these five agonists may occur in nature and one may be follicle-stimulating hormone-releasing hormone. The two peptides with Gln8 and Leu8, if occurring in nature, may have different receptors according to radioreceptor assays and to the ratio of LH/FSH release in vivo. These structures are a basis for the design of antagonists without Arg8 toward avoiding histamine release. Complete inhibition of LH and FSH release in vivo may be induced by joint use of Arg8 and Gln8 or Leu8 antagonists. These potent agonists, related to LHRH, may be therapeutically useful in disorders of reproduction, the central nervous system, and for the control of hormone-dependent carcinomas.
Intrahepatic transport and utilization of biliary glutathione and its metabolites.
Authors: Abbott W A , Meister A
Published: May 2006

Glutathione transported by hepatocytes into the bile canaliculi is metabolized by the actions of gamma-glutamyl transpeptidase and dipeptidase located on the biliary ductular epithelium. This pathway is revealed by the finding of high levels of cyst(e)inylglycine, gamma-glutamylglutathione, gamma-glutamylcyst(e)ine, glutamate, glycine, and cyst(e)ine in bile, by studies in which intrahepatic metabolism of glutathione was inhibited by administration of a potent inhibitor of gamma-glutamyl transpeptidase and by experiments in which glutathione synthesis was inhibited. Canalicular transport of glutathione, as estimated from totals of metabolites found, is much greater than the glutathione found in bile. Glutathione and glutathione metabolites found in bile increase with age, in association with an increase in hepatic glutathione. In younger rats there is apparent uptake of cysteine and glycine moieties that may reflect uptake of cysteinylglycine at the ductular level. This intrahepatic pathway of glutathione transport and metabolism, which resembles that which occurs in the kidney, seems to function as a cellular protective mechanism in the processing of glutathione conjugates and as a recovery system for cysteine moieties.
Gyration is required for 5S RNA transcription from a chromatin template.
Authors: Kmiec E B , Ryoji M , Worcel A
Published: May 2006

We have assembled transcriptionally active chromatin on 5S DNA plasmids by using a Xenopus oocyte supernatant and the 5S-specific transcription factor IIIA (TFIIIA). In this system, the 5S RNA gene is accurately transcribed at a rapid rate of 50 transcripts per gene per hr. By following the time course of RNA synthesis during chromatin assembly, the dose response to TFIIIA addition, and the effect of novobiocin on the assembled nucleoprotein, we show that there is a strict correlation between transcriptional activity and the generation of torsionally strained DNA supercoils in "dynamic chromatin." Transcription cannot be the cause of the dynamic structure, because the assembly of this chromatin is unaffected by alpha-amanitin levels that completely block RNA polymerase III. Surprisingly, the dynamic chromatin remains transcriptionally active after relaxation with DNA topoisomerase I, which implies that the essential parameter for chromatin transcription is gyration per se, and not its effect on DNA topology.
Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution.
Authors: Allured V S , Collier R J , Carroll S F , McKay D B
Published: May 2006

Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.
Induction of c-myc expression in human B lymphocytes by B-cell growth factor and anti-immunoglobulin.
Authors: Lacy J , Sarkar S N , Summers W C
Published: May 2006

Purified human B lymphocytes were examined for transcriptional expression of c-myc in response to mitogenic stimulation by the method of in situ hybridization using 35S-labeled DNA probes. The level of c-myc expression increased 10- to 20-fold within 2 hr after the addition of anti-mu, formalinized Staphylococcus aureus Cowan strain I, or B-cell growth factor, as compared to resting B cells. After 72-96 hr of mitogenic stimulation, c-myc expression remained elevated 5-fold, but expression among individual cells had become more heterogeneous than at early time points. To determine whether c-myc expression in human B lymphocytes is phase specific within the cell cycle, mitogen-stimulated cells were sorted by DNA content into populations of cells in G0/G1, S, and G2/M phases of the cell cycle. Examination of c-myc expression in phase-specific cells revealed that c-myc expression was elevated in all phases of the cell cycle, but it appeared to be maximally expressed in S phase. These studies suggest that c-myc expression in normal human B lymphocytes is cell-cycle dependent and remains elevated in all phases of the cycling cell.
Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells.
Authors: Yamaizumi M , Sugano T , Asahina H , Okada Y , Uchida T
Published: May 2006

Microinjection of cell extracts prepared from both human placenta and HeLa cells into xeroderma pigmentosum (XP) cells of complementation group A restores unscheduled DNA synthesis (UDS) in these cells after UV irradiation [de Jonge, A., Vermeulen, W., Klein, B. & Hoeijmakers, J. (1983) EMBO J. 2, 637-641]. These cells also showed normal resistance to UV irradiation. The half-life of the factors in the cell extracts corresponding to the UDS activity (factor A) was 14 hr in XP cells of group A, and the maximal level of UDS was exerted 2 hr after microinjection. The factors were sensitive to protease treatment but not to RNase treatment and were found to be approximately equal to 160 and approximately equal to 90 kDa by gel filtration. These two fractions of the factor(s) acted specifically in XP cells of complementation group A among complementation groups A, B, C, D, F, G, and probably E and H.
Identification of the binding site on 5S rRNA for the transcription factor IIIA: proposed structure of a common binding site on 5S rRNA and on the gene.
Authors: Huber P W , Wool I G
Published: May 2006

Transcription factor IIIA interacts specifically with an internal control region of Xenopus 5S ribosomal RNA genes and is also a component, along with 5S rRNA, of a 7S ribonucleoprotein particle present in previtellogenic oocytes. We have determined the region of the 5S rRNA in the 7S ribonucleoprotein complex that is protected by the transcription factor from digestion with the ribonuclease alpha-sarcin. The binding site for factor IIIA extends from nucleotide 64 through nucleotide 116; the protected region includes two CCUGG helices separated by 11 nucleotides. The same helices occur in the factor IIIA binding site in the 5S rRNA gene and may constitute a common structural feature recognized by the protein in the gene and in the gene product.
B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.
Authors: Callahan L , Han F S , Watt W , Duchamp D , Kézdy F J , Agarwal K
Published: May 2006

The simulation of the B--Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the RP stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the SP stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral SP oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group of guanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the RP stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the SP stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral SP oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B--Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate oxygens and the N-2 amino group of guanine.
Conservation of extended promoter regions of nodulation genes in
Rhizobium
Authors: Rostas Katalin , Kondorosi Eva , Horvath Beatrix , Simoncsits Andras , Kondorosi Adam
Published: May 2006

A 47-base-pair (bp) conserved sequence in the 5′-flanking regions of three transcriptional units coding for nodulation functions (
nod
ABC,
nod
EFG, and
nod
H) has been identified in
Rhizobium meliloti
strain 41. The conserved region contains subsequences of 7 bp, 5 bp, and 25 bp. The conserved 25-bp sequence was synthesized and used as a hybridization probe; three additional copies of the sequence were identified in
R. meliloti
41; all three were localized in the 135-kb
nod/nif
region of the symbiotic megaplasmid. Nucleotide sequence analysis of the six regions revealed that all contained the 47-bp conserved sequence but, with one exception, adjacent DNA regions did not have long conserved DNA stretches. The position of the 47-bp region was about 200-240 bp upstream of the translational start codons of the three
nod
genes. This conserved sequence is present in several other
Rhizobium
species and located adjacent to
nod
genes. We have demonstrated the involvement of this sequence in the expression of nodulation functions, which suggests that these extended promoter regions may have a role in the coordinated regulation of nodulation genes.
Conservation and diversity in the class I genes of the major histocompatibility complex: sequence analysis of a Tlab gene and comparison with a Tlac gene.
Authors: Pontarotti P A , Mashimo H , Zeff R A , Fisher D A , Hood L , Mellor A , Flavell R A , Nathenson S G
Published: May 2006

The thymus leukemia (TL) antigens, encoded by class I genes in the Tla subregion of the major histocompatibility complex (MHC), are cell surface molecules expressed on thymocytes of certain strains of mice and on certain T-cell leukemias. In order to study the fine structure and interrelationships of genes of the Tla subregion, a Tla-specific probe was isolated from the TL-encoding T13c gene of BALB/c mice (Tlac haplotype). The probe hybridized with two Tla genes in the Tlac haplotype (T13c and T3c) and with only one in the Tlab haplotype (T3b). Examination of this subset of Tla genes (T3b, T3c, and T13c) by restriction enzyme analysis and oligonucleotide hybridization studies confirmed that T3b is the allele of T3c and that T3c and T13c may have arisen by duplication. The T3b gene, while not transcribed in the tissues of the TL- strain C57BL6, was shown to be transcriptionally active in the TL-expressing leukemic cell line ERLD derived from that strain. The T3b gene was cloned and its complete DNA sequence was determined. These data permit complete comparison of two Tla-region genes, T3b and its homologue T13c, and allow us to conclude that these genes show extraordinarily high sequence conservation, in contrast to alleles of the H-2K- and H-2D-region genes. Comparison of T3b with other class I sequences in the H-2 and Qa subregions suggests that the T3-subset genes are the most divergent from other class I genes.
Information transfer between duplicated chromosomal sequences in mammalian cells involves contiguous regions of DNA.
Authors: Liskay R M , Stachelek J L
Published: May 2006

We have investigated the nature of information transfer that appears to occur nonreciprocally between duplicated chromosomal sequences in cultured mouse L cells. We have studied gene conversion between two different defective thymidine kinase genes derived from two closely related strains of type 1 herpes simplex virus and that share a silent restriction site polymorphism. Our results demonstrate that this silent site can be coconverted along with the selected mutant sites. The findings are consistent with a mechanism of gene conversion that involves contiguous blocks of DNA differing in length, position, or both. An additional finding is that the products of coconversion events involving the silent site are unequally recovered although the rates of conversion observed at four different selected sites are similar.
Heat shock locus 93D of Drosophila melanogaster: a spliced RNA most strongly conserved in the intron sequence.
Authors: Garbe J C , Pardue M L
Published: May 2006

The Drosophila melanogaster heat shock locus at 93D encodes at least three overlapping transcripts, 10-12 kilobases (kb), 1.9 kb, and 1.2 kb. The abundance of the three transcripts is significantly increased during heat shock; however, all are also found in non-heat-shocked cells. The 1.2-kb transcript is found in the cytoplasm. Sequence analysis of a 1.1-kb cDNA clone representing sequences within the 1.2-kb transcript and comparison to genomic sequences indicate that it is spliced; 700 base pairs of sequence found in genomic DNA are removed from the middle of the transcript. Sequence analysis further suggests that this RNA does not encode a heat shock protein. The largest open reading frame beginning with a methionine codon would encode a polypeptide of 34 amino acids. We have not been able to detect a heat shock-induced polypeptide of this size. A DNA clone from the analogous heat shock puff of Drosophila hydei has been analyzed by hybridization with the small subclones used to sequence the D. melanogaster cDNA plus a genomic fragment containing the 700-base-pair intron. Results of this hybridization indicated strong homology of the intron fragment. Weaker homology was detected with the two small fragments flanking the intron. Other fragments of the D. melanogaster cDNA showed no hybridization to the cloned D. hydei puff DNA.
Characterization of a murine lymphokine distinct from interleukin 2 and interleukin 3 (IL-3) possessing a T-cell growth factor activity and a mast-cell growth factor activity that synergizes with IL-3.
Authors: Smith C A , Rennick D M
Published: May 2006

Murine mast-cell and T-cell growth factor activities, distinct from interleukins 3 and 2 (IL-3 and IL-2), have been identified and partially purified from the supernatant of the activated helper T-cell line Cl.Ly1+2-/9. This mast-cell growth factor (MCGF) activity supports only low levels of proliferation of several IL-3-dependent mast-cell lines and synergistically enhances the growth of mast cells in the presence of IL-3. The T-cell growth factor (TCGF) stimulates the proliferation of several T-cell lines, but to a lesser extent than recombinant IL-2. The MCGF and TCGF activities were not separable despite multiple biochemical fractionations, suggesting that both activities reside in the same protein. The MCGF/TCGF was separated from endogenous IL-3 by cation-exchange chromatography at neutral pH and could be distinguished from IL-2 by unique elution conditions from reverse-phase columns. Two bands of MCGF/TCGF activity were eluted from gels after sodium dodecyl sulfate/PAGE; under nonreducing conditions, the activities corresponded to molecular masses of 20 and 15 kDa, while after reduction, the molecular masses were 21 and 16 kDa. Thus, both activities may correspond to single polypeptide chains. The majority of the MCGF/TCGF activity appears to reside in the 20-kDa species, which displays a pI of 6.2 on chromatofocusing.
Rearrangements of DNA mediated by terminal transferase.
Authors: Kunkel T A , Gopinathan K P , Dube D K , Snow E T , Loeb L A
Published: May 2006

To assess the involvement of terminal transferase in generating immunoglobulin diversity, the mutagenic potential of this enzyme has been measured. The frequency of single base substitutions during copying of phi X174 DNA by DNA polymerase beta is increased by, at most, 3-fold upon the addition of terminal transferase. However, terminal transferase is highly mutagenic, either alone or with DNA polymerase beta, in a forward mutation assay using M13mp2 DNA. The frequency of complex mutants, as determined by DNA sequence, is increased by greater than 100-fold. These mutants involve the deletion of a variable number of bases initially present in the template sequence and the addition of a sequence of nucleotides rich in guanine residues. Analysis of these mutants suggests an antibody diversity model implicating terminal transferase in the imprecise linkage of variable, joining, and diversity segments during the formation of functional immunoglobulin genes.
Cell cycle-dependent expression of a stable episomal human histone gene in a mouse cell.
Authors: Green L , Schlaffer I , Wright K , Moreno M L , Berand D , Hager G , Stein J , Stein G
Published: May 2006

We have constructed a recombinant plasmid that includes a cell cycle-dependent human H4 histone gene with 650 base pairs of 5' and 900 base pairs of 3' flanking sequences and the 69% transforming fragment of bovine papilloma virus. When transfected into C127 mouse cells, this plasmid is maintained as a stable episome with approximately 20 copies per cell. Micrococcal nuclease digestion indicates that the episomal human histone gene is packaged as chromatin. The human H4 histone transcript is initiated at the correct 5' start site and undergoes selective destabilization when DNA synthesis is inhibited. When C127 cells containing the episomal H4 histone gene are synchronized, the human H4 histone mRNA levels are regulated coordinately with DNA replication and parallel those of transcripts from the murine chromosomal H4 histone genes. Our results suggest that the regulatory sequences and/or regulatory molecules associated with murine and human histone genes are compatible. The human histone gene-bovine papillomavirus episome is therefore a viable system for studying cell cycle-regulated histone gene expression under conditions where control is not influenced at the site of chromosomal integration by cis-acting elements of genes normally not contiguous.
Superoxide dismutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli.
Authors: Eliasson R , Jörnvall H , Reichard P
Published: May 2006

One of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, protein B2, contains in its active form two antiferromagnetically coupled Fe(III) ions and an organic free radical that arises by the one-electron oxidation of tyrosine-122 of the polypeptide chain. Protein B2 lacking the tyrosine radical but with the iron center intact (called protein B2/HU because it is produced by treatment with hydroxyurea) is enzymatically inactive. Previously, it was found that a crude extract from E. coli transforms B2/HU into B2 in the presence of dithiothreitol, Mg2+, and oxygen. On purification of the enzyme system, we now find that radical introduction requires three separate proteins as well as NADPH and FMN. One of the proteins is superoxide dismutase. We hypothesize that the overall reaction involves a reduction of the iron center followed by the oxidation of iron and tyrosine-122. Superoxide dismutase may then be involved in the second step to protect an oxidation-sensitive intermediate. Alternatively, the enzyme might be directly involved in the oxidation step.
Sequences upstream of the STE6 gene required for its expression and regulation by the mating type locus in Saccharomyces cerevisiae.
Authors: Wilson K L , Herskowitz I
Published: May 2006

The STE6 gene of Saccharomyces cerevisiae is an a-specific gene; it is repressed in alpha cells by the alpha 2 product of the mating type locus. To study the role of sequences upstream of STE6 in its regulation and expression, we have determined the DNA sequence of the promoter region, identified the start sites for the STE6 transcript, and identified sequences governing its transcription. Deletions that remove DNA upstream of the STE6 gene were produced and assayed for effects on regulation and expression. The deletions defined two intervals upstream of the STE6 transcription initiation sites. One contains all or part of a negative element; the other contains all or part of a positive element. The negative element is required for repression of STE6 by alpha 2: deletions lacking this element express STE6 constitutively. Such deletions remove a 31-base-pair site, located 135 base pairs upstream of the first transcript start site, that is highly homologous to sites present in the upstream regions of four other genes repressed by alpha 2. These sites are presumably responsible for repression of the a-specific genes by alpha 2. The positive element (a putative upstream activation site) is required for expression of STE6. The deletions define the left boundary of the proposed upstream activation site. Sequence homologies between STE6 and other a-specific genes are found in this region and may mediate activation of this set of genes.
Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids.
Authors: Petit C , Levilliers J , Ott M O , Weiss M C
Published: May 2006

Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators (extinguishers) that prevent the expression of functions foreign to their own differentiation. Here, we report genetic evidence of such control. In microcell hybrids between well-differentiated rat hepatoma cells and microcells of mouse fibroblast L cells, the extinction of albumin synthesis is directly related to the presence of a single specific chromosome of the mouse fibroblast parent. The expression of several other hepatic functions is not affected. Transfection of these hybrids with a recombinant plasmid, containing a tissue-specific control element of the upstream region of the rat albumin gene linked to coding sequences of the chloramphenicol acetyltransferase gene, reveals that extinction acts on or via this cis-control element.
Qa gene expression: biosynthesis and secretion of Qa-2 molecules in activated T cells.
Authors: Soloski M J , Vernachio J , Einhorn G , Lattimore A
Published: May 2006

The biosynthesis and expression of the tissue-specific class I molecule Qa-2 have been studied in resting and activated T-cell populations. Polyclonal activation of T lymphocytes induces a 3- to 4-fold increase in the biosynthesis of Qa-2 molecules but no increase in cell-surface levels. Analysis of the biosynthetic pathway of the Qa-2 molecule in activated lymphocytes reveals that approximately equal to 70% of the newly synthesized Qa-2 molecules are secreted as soluble molecules. In resting-cell populations, Qa-2 remains entirely cell-associated. This process is unique to the Qa-2 molecule, since other class I molecules (e.g., H-2Kb and H-2Db) synthesized by activated cells remain cell-associated. The possibility that the secreted Qa-2 molecule is the product of a new Qa gene or an alternatively spliced mRNA is considered. These results indicate that the Qa-2 molecules may not just function as a cell-surface recognition structure but also may serve a role as a soluble factor synthesized by activated lymphoid cell populations.
Asymmetrical pairings of transposons in and proximal to the white locus of Drosophila account for four classes of regularly occurring exchange products.
Authors: Davis P S , Shen M W , Judd B H
Published: May 2006

An explanation for the origins of four classes of regularly occurring duplication and deficiency chromosomes is provided through examination of their molecular structures. The duplications and deficiencies occur as the reciprocal products of crossing-over, following two different patterns of asymmetrical synapsis between transposons positioned in and proximal to the white locus of Drosophila melanogaster. Three copies of the retrovirus-like transposon roo are involved in the exchanges. Evidence suggests that transposon-mediated asymmetrical exchange is a general phenomenon in eukaryotes, which adds significantly to the effects of transposons in the restructuring of eukaryotic genomes.
Drastically increased expression of MYC and FOS protooncogenes during in vitro differentiation of chronic lymphocytic leukemia cells.
Authors: Larsson L G , Gray H E , Tötterman T , Pettersson U , Nilsson K
Published: May 2006

Chronic lymphocytic leukemia cells, representing a clonal population of resting B lymphocytes, were induced to differentiate into immunoglobulin-secreting lymphoblasts and plasmablasts by phorbol 12-myristate 13-acetate. The induction resulted in a rapid increase in the molar ratio of secreted/membrane-bound mu-chain mRNA. Immunoglobulin secretion was preceded by a transition of the cells from the G0 to G1 phase of the cell cycle, as indicated by an increase in RNA and protein synthesis, and an overall increase in cellular RNA. The cells, however, became blocked in G1 and did not enter S phase. The expression of MYC and FOS was rapidly induced by the phorbol 12-myristate 13-acetate treatment. The induction of FOS preceded the shift in secreted/membrane-bound mu-chain mRNA molar ratio, while that of MYC occurred concomitantly with the shift, but prior to induction of total RNA synthesis and immunoglobulin secretion. MYC expression remained at a relatively high level during the whole differentiation process. It is thus concluded that a decline of MYC expression is not a prerequisite for differentiation of the chronic lymphocytic leukemia cells. This suggests that MYC expression may play a different role during differentiation of nonproliferating B cells than in the myelomonocytic cell lines HL-60 and U-937, where MYC expression has been reported to decrease during induced differentiation. The results also show that the expression of the MYC and FOS genes does not result in the transition of these cells into the S phase of the cell cycle.
NH2-terminal sequences of two src proteins that cause aberrant transformation.
Authors: Garber E A , Hanafusa H
Published: May 2006

Two isolates of recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, transform cells in culture, but have greatly reduced in vivo tumorigenicity. The src proteins of rASV157 and rASV1702 have alterations within their NH2 termini, are not myristoylated, and have an altered subcellular localization. We have molecularly cloned and determined the nucleotide sequences of the src genes of rASV157 and rASV1702. We found that their src proteins have unusual NH2 termini: the rASV157 src protein NH2 terminus consists of 30 amino acids of the env signal peptide attached to Ser-6 of the src sequence, while the rASV1702 src protein NH2 terminus consists of 45 amino acids of the env signal peptide attached to Ala-76 of the src sequence. Expression of recombinant Rous sarcoma virus constructs containing the molecularly cloned rASV src genes produced src proteins with the same properties as those of the parental viruses. Our results suggest that the NH2-terminal structures are responsible for many unusual properties of the mutant src proteins.
Differential mRNA transcription during salinity stress in barley
Authors: Ramagopal Subbanaidu
Published: May 2006

The molecular and genetic bases of salinity tolerance in plants are not understood. Gene expression at the mRNA level was investigated in a salt-tolerant and a salt-sensitive genotype of barley. Seedlings were exposed to NaCl stress and translatable mRNAs were isolated from root and shoot tissues. A reticulocyte cell-free system was programed with barley mRNAs and the
in vitro
products were resolved on two-dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. The functional mRNAs in unstressed seedlings were qualitatively almost indistinguishable in the two genotypes. However, salinity stress triggered differential transcription of specific mRNAs depending upon genotype and tissue. In roots, 12 new mRNAs were induced that encoded proteins of 21-34 kDa, with a pI range of 6.1-7.7. In shoots, the 9 new mRNAs coded for proteins of 18-50.5 kDa, with a pI range of 5.4-7.8. These new stress mRNAs represented one of two main classes. Class I consisted of mRNAs shared by both genotypes. Class II represented mRNAs specific to each genotype; unique mRNAs of roots accumulated preferentially in the salt-tolerant genotype, whereas those of shoots accumulated in the salt-sensitive genotype. The findings suggest that transcriptional as well as posttranscriptional mechanisms regulate gene expression in barley during salinity stress.
Phytotoxins from the pathogenic fungi
Drechslera maydis
and
Drechslera sorghicola
Authors: Sugawara F. , Strobel G. , Strange R. N. , Siedow J. N. , Van Duyne G. D. , Clardy J.
Published: May 2006

Drechslera maydis
, the causal agent of Southern corn leaf blight, and
Drechslera sorghicola
, the causal agent of leaf spot on Johnson grass, produce a series of phytotoxic sesterterpenoids. These sesterterpenoids belong to the ophiobolin family. One of them, ophiobolin I, was characterized by x-ray diffraction and served as a crucial reference compound for characterizing four other ophiobolins. All of the ophiobolins studied produce characteristic lesions on host plants at concentrations of 1 mM to 1 μM. The ophiobolin characterized as 6-epiophiobolin A is selectively toxic to corn bearing Texas-male-sterile (Tms) cytoplasm when assayed in a dark CO
2
fixation assay. It is plausible that these ophiobolins had a role in the 1970 corn-blight epidemic in North America.
Periodic crosslinking of microtubules by cytoplasmic microtubule-associated and microtubule-corset proteins from a trypanosomatid.
Authors: Bramblett G T , Chang S L , Flavin M
Published: May 2006

The dominant element in the cytoskeleton of Crithidia fasciculata is a peripheral corset of microtubules enclosing the cell body and closely underlying the plasma membrane. A lateral spacing of 50 nm is maintained by crosslinks, and microtubules may also be linked to the plasma membrane. We have characterized groups of polypeptides that associate with microtubules polymerized in vitro from the cytoplasm, or that are associated with the corset complex. They differ except for one of Mr 33,000 present in both groups. The corresponding native corset protein appears to be a dimer of Mr 66,000. These protein(s) copolymerize with brain tubulin, and the resultant polymer consists of pairs or small parallel bundles of microtubules, joined by periodic crosslinks spaced about 8.5 nm apart.
Functional epithelial cell line cloned from rat parathyroid glands.
Authors: Sakaguchi K , Santora A , Zimering M , Curcio F , Aurbach G D , Brandi M L
Published: May 2006

Primary cultures of rat parathyroid cells were developed in medium containing 5% calf serum, 1% Nutridoma-SP (a serum-free medium supplement from Boehringer Mannheim), and 0.7 mM calcium. The PT-r strain was purified by successive colony isolations and maintained differentiated characteristics (secretion of bioactive and radioimmunoactive parathyroid hormone into the culture medium, sensitivity to calcium regulation, and modulation by secretin) for 7 months in continuous culture. These cultured cells are epithelioid, display diploid chromosome numbers, and do not show a transformed phenotype. There has been no decrease in the rate of cell division or decline in parathyroid hormone secretion since the cell line was established. This clonal cell line provides an important system for further studies on the biology of the parathyroid cell.
Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal human bronchial epithelial cells.
Authors: Wilson V L , Smith R A , Longoria J , Liotta M A , Harper C M , Harris C C
Published: May 2006

The genomic content of DNA 5-methyldeoxycytidine (m5dC) was measured in dividing normal human bronchial epithelial cells treated with a broad range of chemical carcinogens. At noncytotoxic concentrations, all of the carcinogenic agents tested significantly reduced cellular DNA m5dC content whereas the weakly carcinogenic and noncarcinogenic agents, benzo[e]pyrene and phenanthrene (respectively), did not. These reductions varied from 8% to 31% depending on the agent and the donor cells. The reductions in genomic m5dC levels were concentration dependent for the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene. We speculate that carcinogen-induced perturbation of DNA m5dC patterns may lead to heritable changes in gene expression and contribute to the molecular alterations involved in the initiation and the subsequent steps of the carcinogenesis process.
Regulation of murine class I genes by interferons is controlled by regions located both 5' and 3' to the transcription initiation site.
Authors: Korber B , Hood L , Stroynowski I
Published: May 2006

Interferons regulate the expression of a large number of mammalian genes, including the major histocompatibility antigen genes. To investigate the mechanisms involved in interferon action, we have analyzed the ability of murine H-2Ld and H-2Dd DNA sequences to control the responses to interferon. The results indicate that interferon regulation of class I gene expression is complex and involves at least two mechanisms that are dependent on class I sequences located upstream and downstream to the transcription initiation site. In transfected mouse L cells, both of these regions are required for full enhancement of class I gene expression, with the major portion of the response controlled by the sequences located 3' to the transcription initiation site. The fine-mapping analysis of the 5' region-encoded response also suggests that recombinant alpha and gamma interferons may exert their effects on class I gene expression by using different cis-acting regulatory sequences.
Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23.
Authors: Wang F , Gregory C D , Rowe M , Rickinson A B , Wang D , Birkenbach M , Kikutani H , Kishimoto T , Kieff E
Published: May 2006

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells induces some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line, including BL markers (cALLA and BLA), proliferation markers (transferrin receptor and BK19.9), and cell adhesion-related molecules (LFA-1 and LFA-3). Increased CD23 expression in cells expressing EBNA-2 was apparent from monoclonal anti-CD23 antibody binding to the cell surface, from immunoprecipitation of the 45-kDa and 90-kDa CD23 proteins with monoclonal antibody, and from RNA blots probed with labeled CD23 DNA. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.
Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP.
Authors: Lewis E J , Harrington C A , Chikaraishi D M
Published: May 2006

Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to +27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained within 773 bases.
Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate.
Authors: Pontremoli S , Melloni E , Michetti M , Sparatore B , Salamino F , Sacco O , Horecker B L
Published: May 2006

Stimulation of intact human neutrophils with phorbol 12-myristate 13-acetate results in the selective phosphorylation of two cytoskeletal protein components with molecular masses of 20 and 48 kDa. After phosphorylation the 48-kDa protein is no longer recovered as a component of the cytoskeletal fraction but is present as a fully soluble phosphoprotein. Phosphorylation of the 20-kDa protein (probably myosin light chains) signals a proteolytic conversion, catalyzed by calpain, to a smaller species having a molecular mass of approximately 15 kDa. Phosphorylation of both the 48- and 20-kDa proteins is related to the conversion of protein kinase C, also catalyzed by calpain, to the soluble fully active form. Leupeptin, an inhibitor of calpain, blocks both the phosphorylation of the target proteins and the proteolytic modification of the 20-kDa polypeptide. Thus, phosphorylation of cytoskeletal proteins and signal-directed proteolysis appear to be related processes that follow stimulation of human neutrophils by phorbol esters. The resulting changes in cytoskeletal organization may be involved in the expression of some neutrophil functions, such as exocytosis of specific granules.