bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of free fatty acids on the organization of cytoskeletal elements in lymphocytes.
Authors: Hoover R L , Fujiwara K , Klausner R D , Bhalla D K , Tucker R , Karnovsky M J
Published: September 2015

Treatment of mouse lymphocytes with cis-unsaturated free fatty acids produced alterations in the immunofluorescence patterns of the cytoskeleton and contractile proteins. Saturated free fatty acids and trans-unsaturated free fatty acids had no effect. In untreated cells, the microtubular pattern exhibited radiation from an organizing center, resembling the spokes of an umbrella. The addition of linoleic acid produced a polarized submembranous aggregate. Under control conditions, staining for actin revealed a diffuse pattern over the entire cell, but the addition of linoleic acid caused the formation of a single large patch, or polarized submembranous aggregate. The pattern for alpha-actinin normally revealed intense perinuclear staining on a diffuse background. Linoleic acid caused the loss of this pattern and the formation of a polarized submembranous aggregate. Linoleic acid treatment also caused the pattern for myosin to change from diffuse to uniform submembranous patching around the periphery of the cell. For all of these proteins, calcium (8 mM), but not magnesium, partially reversed the effects of linoleic acid. Sodium azide had little effect on the normal distribution of actin, tubulin, and alpha-actinin; however, myosin staining revealed prominent patch formation. Colchicine treatment caused diffuse staining, some polarized submembranous aggregate formation of tubulin, and some patching of myosin, but not as extensively as did treatment with linoleic acid. Actin and alpha-actinin were unaffected. These results, in view of the previously shown facts that pretreatment of cells with linoleic acid followed by anti-immunoglobulin inhibits capping of surface immunoglobulin (Klausner, et al., Proc. Natl. Acad. Sci. U.S.A. 77:437-441, 1980) and that free fatty acids partition into the surface membrane (Klausner et al., J. Biol. Chem. 255:1286-1295, 1980), suggest that the perturbation of the plasma membrane with unsaturated free fatty acids alters the interaction of surface receptors with the cytoskeleton, which in turn affects cytoplasmic distribution of the proteins.
Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase.
Authors: Veenhuis M , Harder W , van Dijken J P , Mayer F
Published: September 2015

The substructural organization of completely crystalline peroxisomes present in Hansenula polymorpha cells grown under methanol limitation in a chemostat was investigated by different cytochemical and ultrastructural techniques. Time-dependent cytochemical staining experiments indicated that activities of the two main constituents of these organelles, namely, alcohol oxidase and catalase, were present throughout the crystalline matrix. Catalase was completely removed from isolated peroxisomes by osmotic shock treatment. After such treatment, the ultrastructure of the crystalline matrix of the organelles remained virtually intact. Because alcohol oxidase activity was still present in this matrix, it was concluded that alcohol oxidase protein is the only structural element of the peroxisomal crystalloids. The molecular architecture of the crystalloids was investigated in ultrathin cryosections which permitted recognition of individual molecules in the crystalline matrix. Depending on the plane of sectioning, different crystalline patterns were observed. Tilting experiments indicated that these images were caused by superposition of octameric alcohol oxidase molecules arranged in a tetragonal lattice. A three-dimensional model of the crystalloid is presented. The repeating unit of this structure is composed of four alcohol oxidase molecules. The crystalloid represents an open structure, which may explain the observed free mobility of catalase molecules.
rme1 Mutation of Saccharomyces cerevisiae: map position and bypass of mating type locus control of sporulation.
Authors: Rine J , Sprague G F , Herskowitz I
Published: September 2015

Sporulation in Saccharomyces cerevisiae normally occurs only in MATa/MAT alpha diploids. We show that mutations in RME1 bypassed the requirements for both a and alpha mating type information in sporulation and therefore allowed MATa/MATa and MAT alpha/MAT alpha diploids to sporulate. RME1 was located on chromosome VII, between LEU1 and ADE6.
Transposition of yeast mating type genes from two translocations of the left arm of chromosome III.
Authors: Haber J E , Rowe L , Rogers D T
Published: September 2015

In the yeast Saccharomyces cerevisiae, the HIS4C gene lies on the left arm of chromosome III. We analyzed two chromosomal rearrangements that have HIS4C translocated either to chromosome XII or to a new translocation chromosome. Using the cmt mutation that allows expression of the normally silent copies of mating type genes, we found that both of these translocations also carried HML alpha, more than 30 map units distal to HIS4C which normally lies on chromosome III. In the case of the translocation chromosome (designated T3), we also found an exchange event between HML alpha on the translocation chromosome and HMLa on chromosome III. In diploids containing two T3 chromosomes (one carrying HML alpha and the carrying HMLa), we found that HML was 32 centimorgans from HIS4C, which was 10 centimorgans from an unknown centromere. In homothallic strains carrying HMLa MATa HMRa on chromosome III, switching from MATa to MAT alpha could occur by using the HML alpha on the translocation as the sole donor of alpha information. Transposition from HML alpha on chromosome T3 was about 20 to 40% as efficient as transposition from intact chromosome III. In contrast, transposition from the HML alpha inserted into chromosome XII was reduced about 100-fold. This reduced efficiency did not appear to be caused by an alteration in the sequences immediately surrounding HML alpha in the translocation. The translocated HML alpha sequence was located in the same size (29-kilobase) SalI fragment as was found in chromosome III, and the same EcoRI, HindIII, and BglII restriction sites were also found. Furthermore, HML alpha was still under the control of the CMT gene, which maintains HML as a silent copy of mating type information. These results suggested that the position of the HML alpha sequence plays an important role in the efficiency of mating type switching.
Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.
Authors: Alexandraki D , Ruderman J V
Published: September 2015

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.
Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.
Authors: Hendrick J P , Wolin S L , Rinke J , Lerner M R , Steitz J A
Published: September 2015

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.
Membrane protein redistribution during differentiation of cultured human erythroleukemic cells.
Authors: Hunt R C , Marshall L M
Published: September 2015

Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.
Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum.
Authors: Clark J M , Deering R A
Published: September 2015

A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostelium discoideum. Analysis of the frequency of T4 endonuclease V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (rad+) removed greater than 98% of the dimers induced by irradiation at 40 J/m2 (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8% were removed during the 10- to 15-min period immediately after irradiation, whereas NC4 excised 64% during this interval. HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic acid at the sites of pyrimidine dimers.
Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing.
Authors: Harpold M M , Wilson M C , Darnell J E
Published: September 2015

We have further analyzed the metabolism of specific messenger ribonucleic acid (mRNA) sequences within the cytoplasmic and nuclear RNA of Chinese hamster ovary (CHO) cells by using a set of previously constructed complementary deoxyribonucleic acid (DNA) clones (Harpold et al., Cell 17:1025-1035, 1979) as specific molecular probes in a variety of RNA:DNA hybridization experiments. The majority of the labeled mRNA complementary to each of the nine clones was found in the polyribosomes, with some variation between individual sequences. The great majority of each specific mRNA labeled for 3 h or less was in the polyadenylated [poly(A)+] fraction. However, the amount of each sequence increased in the non-poly(A)+ [poly(A)-] fraction after very long label times, suggesting the derivation of the poly(A)- RNA from the poly(A)+ RNA. Eight of the nine mRNA's have cytoplasmic half-lives ranging from 8 to 14 h, whereas one of the mRNA's, the scarcest in the group, has a somewhat shorter half-life of approximately 3 h. The proportion of each of the specific long-lived mRNA's within the total labeled mRNA increased as a function of labeling time, indicating that a large fraction, probably greater than 50%, of the initially labeled poly(A)+ mRNA in CHO cells has a half-life of less than 3 h. A quantitative analysis of the kinetics of labeling of specific nuclear and cytoplasmic sequences indicated that a significant fraction of the mRNA sequences transcribed from genes containing these nine CHO sequences were successfully processed into mRNA. However, two of the CHO mRNA sequences were only partially conserved during nuclear processing to yield mRNA. These studies demonstrated that events at two post-transcriptional levels, differential nuclear processing efficiency of different primary transcripts and cytoplasmic stability of different mRNA's, can be involved in the determination of the cytoplasmic concentrations of different mRNA's.
Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors.
Authors: Otsuka A , de Paolis A , Tocchini-Valentini G P
Published: September 2015

A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences.
Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.
Authors: Doersen C J , Stanbridge E J
Published: September 2015

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.
Tumor promoters alter the temporal program of adenovirus replication in human cells.
Authors: Fisher P B , Young C S , Weinstein I B , Carter T H
Published: September 2015

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.
Linear mitochondrial deoxyribonucleic acid from the yeast Hansenula mrakii.
Authors: Wesolowski M , Fukuhara H
Published: September 2015

The mitochondrial deoxyribonucleic acid (mtDNA) from a petite-negative yeast, Hansenula mrakii, was studied. A linear restriction map was constructed with 11 restriction enzymes. The linearity of the genome was confirmed by direct end labeling of the molecule, followed by restriction analysis. The molecular weight of the DNA was found to be 55,000 base pairs. This is the first linear mtDNA found in yeast species. Using specific gene probes obtained from Saccharomyces cerevisiae mtDNA, we have constructed a gene map of H. mrakii mtDNA. The arrangement of genes in this linear genome was very different from the circular mtDNA of other known yeasts.
Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.
Authors: Adelman S F , Howett M K , Rapp F
Published: September 2015

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.
Number and size of human X chromosome fragments transferred to mouse cells by chromosome-mediated gene transfer.
Authors: Olsen A S , McBride O W , Moore D E
Published: September 2015

Labeled probes of unique-sequence human X chromosomal deoxyribonucleic acid, prepared by two different procedures, were used to measure the amount of human X chromosomal deoxyribonucleic acid in 12 mouse cell lines expressing human hypoxanthine phosphoribosyltransferase after chromosome-mediated gene transfer. The amount of X chromosomal deoxyribonucleic acid detected by this procedure ranged from undetectable levels in the three stable transformants and some unstable transformants examined to about 20% of the human X chromosome in two unstable transformants. Reassociation kinetics of the X chromosomal probe with deoxyribonucleic acid from the two unstable transformants containing 15 to 20% of the human X chromosome indicate that a single copy of these sequences is present. In one of these lines, the X chromosomal sequences exist as multiple fragments which were not concordantly segregated when the cells were selected for loss of hprt.
Factors governing the expression of a bacterial gene in mammalian cells.
Authors: Mulligan R C , Berg P
Published: September 2015

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.
Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.
Authors: Sarver N , Gruss P , Law M F , Khoury G , Howley P M
Published: September 2015

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.
Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.
Authors: Weiffenbach B , Haber J E
Published: September 2015

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.
Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae.
Authors: Kiss G B , Amin A A , Pearlman R E
Published: September 2015

Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator.
Internalization of ricin in Chinese hamster ovary cells.
Authors: Ray B , Wu H C
Published: September 2015

Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells.
The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element.
Authors: Haynes S R , Toomey T P , Leinwand L , Jelinek W R
Published: September 2015

A consensus sequence has been determined for a major interspersed deoxyribonucleic acid repeat in the genome of Chinese hamster ovary cells (CHO cells). This sequence is extensively homologous to (i) the human Alu sequence (P. L. Deininger et al., J. Mol. Biol., in press), (ii) the mouse B1 interspersed repetitious sequence (Krayev et al., Nucleic Acids Res. 8:1201-1215, 1980) (iii) an interspersed repetitious sequence from African green monkey deoxyribonucleic acid (Dhruva et al., Proc. Natl. Acad. Sci. U.S.A. 77:4514-4518, 1980) and (iv) the CHO and mouse 4.5S ribonucleic acid (this report; F. Harada and N. Kato, Nucleic Acids Res. 8:1273-1285, 1980). Because the CHO consensus sequence shows significant homology to the human Alu sequence it is termed the CHO Alu-equivalent sequence. A conserved structure surrounding CHO Alu-equivalent family members can be recognized. It is similar to that surrounding the human Alu and the mouse B1 sequences, and is represented as follows: direct repeat-CHO-Alu-A-rich sequence-direct repeat. A composite interspersed repetitious sequence has been identified. Its structure is represented as follows: direct repeat-residue 47 to 107 of CHO-Alu-non-Alu repetitious sequence-A-rich sequence-direct repeat. Because the Alu flanking sequences resemble those that flank known transposable elements, we think it likely that the Alu sequence dispersed throughout the mammalian genome by transposition.
Organization and expression of multiple actin genes in the sea urchin.
Authors: Scheller R H , McAllister L B , Crain W R , Durica D S , Posakony J W , Thomas T L , Britten R J , Davidson E H
Published: September 2015

A set of at least 11 actin genes has been isolated from genomic recombinant deoxyribonucleic acid libraries of the sea urchin Strongylocentrotus purpuratus. Most of the isolates derive from a library which represents the genome of a single animal. There are at least five distinct types of sea urchin actin gene, some of which are represented by multiple copies in the genome. The actin gene types are distinguished by nonhomologous flanking sequences and intervening sequences, though the protein coding sequences appear in most cases to be quite similar. Eight of the 11 genes isolated have been recovered in lambda recombinants that contain two actin genes, linked at 5- to 9-kilobase distances. Restriction map overlaps suggest that the genome contains an array of at least three of these genes spaced over about 30 kilobases of deoxyribonucleic acid. In the linkage patterns observed, actin genes of diverse types were linked to each other. In early embryos, actin messenger ribonucleic acid (RNA) transcripts of 1.8 and 2.2 kilobases were found, and the longer of these transcripts was more prevalent in the maternal RNA of the egg. From RNA gel blot experiments, we conclude that the two transcripts derive from different actin gene types. Different repetitive sequences were located to either side of most of the actin genes, and in most observed cases the repeat sequences which were adjacent to actin genes of a given type were similar. The repeat sequences flanking the actin genes belonged to families which were transcribed, but those repeats in the neighborhood of the actin genes which have been investigated were not themselves represented in the stable RNAs of eggs or early embryos.
Assignment of the chloramphenicol resistance gene to mitochondrial deoxyribonucleic acid and analysis of its expression in cultured human cells.
Authors: Wallace D C
Published: September 2015

The mitochondrial deoxyribonucleic acids (mtDNA's) from human HeLa and HT1080 cells differed in their restriction endonuclease cleavage patterns for HaeII, HaeIII, and HhaI. HaeII digestion yielded a 9-kilobase fragment in HT1080, which was replaced by 4.5-, 2.4-, and 2.1-kilobase fragments in HeLa. HaeIII and HhaI yielded distinctive 1.35- and 0.68-kilobase HeLa fragments. These restriction endonuclease polymorphisms were used as mtDNA markers in HeLa-HT1080 cybrid and hybrid crosses involving the cytoplasmic chloramphenicol resistance mutation was used. mtDNA's were purified and digested with the restriction endonucleases, the fragments were separated on agarose gels, and the bands were detected by ethidium bromide staining and Southern transfer analysis. Three cybrids and four hybrids (four expressing HeLa and three expressing HT1080 chloramphenicol resistance) contained 2- to 10-fold excesses of the mtDNA of the chloramphenicol-resistant parent. One cybrid, which was permitted to segregate chloramphenicol resistance and was then rechallenged with chloramphenicol, had approximately equal proportions of the two mtDNA's. Only one hybrid was discordant. These results indicated that chloramphenicol resistance is encoded in mtDNA and that expression of chloramphenicol resistance is related to the ratio of chloramphenicol-resistant and -sensitive genomes within cells.
High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.
Authors: Sandri-Goldin R M , Goldin A L , Levine M , Glorioso J C
Published: September 2015

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.
Linkage and evolutionary diversification of developmentally regulated multigene families: tandem arrays of the 401/18 chorion gene pair in silkmoths.
Authors: Jones C W , Kafatos F C
Published: September 2015

The coordinately expressed silkmoth chorion genes, 401 and 18, are closely linked as a pair, in divergent orientation. Analysis of overlapping clones (chromosomal "walk") demonstrated that each of the multiple copies of this gene pair is embedded within a larger deoxyribonucleic acid unit, which is tandemly repeated in a few arrays or possibly a single array. Southern analysis and examination of clones from a single individual moth demonstrated that the repeat units are extensively polymorphic in restriction sites, length, and possibly number, no differential amplification was evident during choriogenesis. Intron and 5'-flanking sequences were shown to be specific for the 401/18 gene pair and not to be present elsewhere in the genome. The spatial distribution of variations in the genes and their flanking sequences were examined.
Ascorbate induction of collagen synthesis as a means for elucidating a mechanism of quantitative control of tissue-specific function.
Authors: Schwarz R I , Mandell R B , Bissell M J
Published: September 2015

Ascorbic acid displays the characteristics of an ideal inducer of tissue-specific function in primary avian tendon cells in culture. It is a highly specific, potent stimulator of collagen synthesis, it demonstrates slow reversible kinetics, and it has no effect on growth rate of the cultured cells. Kinetic analysis of ascorbate induction of collagen synthesis was used to determine the critical steps in this complex biosynthetic pathway. Full hydroxylation of the proline residues in collagen, although probably a necessary step for collagen induction, was in itself not sufficient for achieving either increased secretion or increased synthesis. On the other hand, an increase in secretion rate, which required both the presence of ascorbate and a high cell density, did correlate with the later stimulation in procollagen production. The process of procollagen secretion, therefore, meets the minimal requirements for the rate-limiting step. The fact that the cells maintained a large pool of intracellular procollagen despite changes in the rates of translation or secretion led us to postulate a possible feedback between the level of the internal procollagen pool and the rate of procollagen synthesis.
Cellular Moloney murine sarcoma (c-mos) sequences are hypermethylated and transcriptionally silent in normal and transformed rodent cells.
Authors: Gattoni S , Kirschmeier P , Weinstein I B , Escobedo J , Dina D
Published: September 2015

Moloney murine sarcoma virus carries an oncogenic sequence (v-mos) which is homologous to a single copy gene (c-mos) present in the normal cells of several vertebrate species. Because of the possible significance of c-mos sequences in normal development and malignant transformation induced by physical or chemical agents, we have examined the state of integration, methylation, and transcriptional activity of c-mos sequences in a variety of normal rodent tissues, normal cell lines, or cell lines transformed by radiation or chemical carcinogens. DNA-DNA hybridization, utilizing the Southern blotting technique and a plasmid-derived DNA probe representing the v-mos sequence, gave no evidence for rearrangements of the c-mos sequence in the DNAs obtained from these diverse cell types. Parallel studies employing the restriction enzyme isoschizomers HpaII and MspI indicated that in all of these cell types the c-mos sequences were heavily methylated. In addition, analysis of cellular RNAs by blot hybridization with the v-mos probe failed to detect evidence of transcription of the c-mos sequences in any of these cell types. This was in contrast to a Moloney sarcoma virus-transformed cell line in which we found that the integrated v-mos sequence was both undermethylated and extensively transcribed. Thus, it would appear that c-mos sequences do not play a role in the transformation of rodent cells by chemical or physical agents, although the possible role of other endogenous onc sequences remains to be determined.
Isolation and subcloning analysis of functional centromere DNA (CEN11) from Saccharomyces cerevisiae chromosome XI.
Authors: Fitzgerald-Hayes M , Buhler J M , Cooper T G , Carbon J
Published: September 2015

We have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Genetic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also contained on the segment of S. cerevisiae DNA cloned in pYe(MET14)2. This plasmid is maintained stably in budding S. cerevisiae cultures and segregates predominantly 2+:20- through meiosis. The CEN11 element has been subcloned in vector YRp7' on an S. cerevisiae DNA fragment 900 base pairs in length [pYe(CEN11)10]. The mitotic and meiotic behavior of plasmids containing CEN11 plus a DNA replicator (ars) indicates that the centromere DNA sequences enable these plasmids to function as true minichromosomes in S. cerevisiae.
Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides.
Authors: Kinlaw C S , Dusing-Swartz S K , Berget S M
Published: September 2015

Immunoprecipitation of human small nuclear ribonucleoproteins (snRNPs) containing the small nuclear RNAs U1, U2, U4, U5, and U6 with two antibodies produced in certain patients suffering from systemic lupus erythematosus was used to identify the polypeptides present on human U1 and U2 snRNPs. U1 and U2 snRNPs contain both common and unique polypeptides; visualization of the differences was possible through the use of non-methionine protein labeling and partial fractionation of snRNP populations. To facilitate comparisons with results from other laboratories, we have designated the snRNP polypeptides by their molecular weights. Four small polypeptides, P8, P9, P10, and P12, of 8,000 to 12,000 daltons, are each present in equal amounts on both U1 and U2 snRNPs. U1 snRNPs also contain a unique 30,000-dalton polypeptide, P30, whereas U2 snRNPs contain a unique 27,000-dalton, methionine-deficient polypeptide, P27. A closely migrating pair of polypeptides, P23 and P22, of 23,000 and 21,500 daltons, respectively, is present on both snRNPs; U2 snRNPs are enriched in the former, and U1 snRNPs are enriched in the latter.
Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.
Authors: Perlman J , Feldman J F
Published: September 2015

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.
Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.
Authors: Silverman S J , Rose M , Botstein D , Fink G R
Published: September 2015

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.
Lectin-resistant mutants of polarized epithelial cells.
Authors: Meiss H K , Green R F , Rodriguez-Boulan E J
Published: September 2015

Two lectin-resistant mutants derived from Madin Darby canine kidney cells, with constitutive alterations in the asparagine-linked carbohydrate moieties, retained the characteristic structural and functional epithelial polarity of the parental cells. A ricin-resistant cell line was unable to incorporate galactose-sialic acid into glycoproteins and, from the pattern of cross-resistance to other lectins, appears to be different from previously described lines resistant to this lectin: the mutation in a concanavalin A-resistant line results, probably, in the production of defective carbohydrate cores of glycoproteins. In spite of glycosylation defects which result in an increased electrophoretic mobility of many cellular glycoproteins, both mutants retained the typical asymmetric structure of the plasma membrane (microvilli on the apical surface, junctional elements on the basolateral surface), functional tight junctions, and unidirectional active transport of electrolytes and water. These results suggest that glycoproteins with terminal galactose-sialic acid moieties are not critically involved in the development and maintenance of polarity in epithelial cells. The mutant cells, particularly the ricin-resistant line, exhibited, however, morphological and electrophysiological changes which suggest a quantitative effect of the mutations on intracellular traffic of membranes and tight junction formation. The cell lines described in this paper, the first lectin-resistant mutants of epithelial lineage, should prove useful tools for studying the peculiarities of glycosylating pathways in polarized cells.
Propranolol, atenolol, and trifluoperazine reduce the spontaneous occurrence of meiotic diploid products in Saccharomyces cerevisiae.
Authors: Sora S , Bianchi M
Published: September 2015

The effect of atenolol, propranolol, trifluoperazine, and caffeine on the occurrence of meiotic diploid and disomic products in Saccharomyces cerevisiae was investigated. We demonstrated that atenolol, propranolol, and trifluoperazine reduce the occurrence of meiotic diploid products and that propranolol also slightly decreases the spontaneous frequency of disomics. On the other hand, caffeine appears to be a powerful inducer of diploid meiotic products, but also shows a lesser effect on disomic induction. Since spontaneous or caffeine-induced diploids arise from a failure of the second meiotic division, it appears that the target of these drugs is at the beginning of the second meiotic division. The only common effect of trifluoperazine and propranolol, mainly investigated in mammals, was an inhibition of calmodulin activity via direct interaction. We tend, therefore, to believe that calmodulin activity must be a crucial point for the second meiotic division to begin. The increased induction of diploids, due to caffeine, may be interpreted as a consequence of an increased cyclic AMP level.