bluuebunny/crossref_metadata_2025 huggingface dataset:
55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.
Authors: Scheinberg D A , Strand M
Published: September 2015

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.
Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.
Authors: Dunn-Coleman N S , Robey E A , Tomsett A B , Garrett R H
Published: September 2015

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.
Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae.
Authors: Matsumoto K , Toh-e A , Oshima Y
Published: September 2015

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.
Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum.
Authors: Charlang G , Ng B , Horowitz N H , Horowitz R M
Published: September 2015

Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.
Heat-sensitive mutant strain of Neurospora crassa, 4M(t), conditionally defective in 25S ribosomal ribonucleic acid production.
Authors: Loo M W , Schricker N S , Russell P J
Published: September 2015

A heat-sensitive mutant strain of Neurospora crassa, 4M(t), was studied in an attempt to define its molecular lesion. The mutant strain is inhibited in conidial germination and mycelial extension at the nonpermissive temperature (37 degrees C). Macromolecular synthesis studies showed that both ribonucleic acid (RNA) and protein syntheses are inhibited when 4-h cultures are shifted from 20 to 37 degrees C. Density gradient analysis of ribosomal subunits made at 37 degrees C indicated that strain 4M(t) is deficient in the accumulation of 60S ribosomal subunits in that the ratio of 60S/37S subunits was 0.29:1 compared with 1.6:1 for the parental strain. This phenotype was shown to be the result of a slow rate of processing of, and a deficiency in the amount of, the immediate precursor to 25S ribosomal RNA (the large RNA of the 60S subunit) in the sequence of events constituting the production of mature ribosomal RNAs from the primary transcript of the ribosomal deoxyribonucleic acid, the precursor ribosomal RNA molecule. Analysis of polysomes suggested that the heat-sensitive gene product might function in both the assembly and the function of the 60S ribosomal subunit, since there was a smaller proportion of newly made 60S subunits synthesized at 37 degrees C in the polysome region of the gradients than in the monosome-plus-subunit region. The ribosomal RNA processing defect is apparently responsible for the observed defects in germination and macromolecular synthesis at 37 degrees C, but the precise molecular lesion is not known. On the basis of these results, the heat-sensitive mutant allele in the 4M(t) strain is considered to define the rip1 (ribosome production) gene locus.
Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae.
Authors: Shalit P , Loughney K , Olson M V , Hall B D
Published: September 2015

CYC1 and sup4 are part of a tightly linked cluster of genes on chromosome X in the yeast Saccharomyces cerevisiae. Using as probes previously cloned fragments containing the CYC1 and sup4 genes, we have identified and cloned the deoxyribonucleic acid (DNA) present between these genes in one strain of yeast. We find that the CYC1 and sup4 genes are approximately 21 kilobases apart. In the same strain, the meiotic map distance is approximately 3.7 centimorgans, for a ratio of 5.6 kilobases per centimorgan in this interval. The physical mapping has allowed unambiguous determination of the orientation of CYC1 and sup4 relative to each other, the centromere, and a nearby transfer ribonucleic acid (tRNA(2Ser)) gene. The spontaneous mutation cyc1-1 inactivates the CYC1 gene as well as the neighboring loci OSM1 and RAD7. We have determined that a cyc1-1-bearing strain lacks approximately 13 kilobases of single-copy DNA from the CYC1-sup4 region, including all of the CYC1 coding information. There is a sequence homologous to the middle-repetitive element Ty1 at or near the breakpoint of the cyc1-1 deletion. We discuss the possibility that Ty elements play a role in the formation of such large, spontaneous deletions, which occur frequently in this region of chromosome X in certain yeast strains.
Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.
Authors: Yarosh D B , Setlow R B
Published: September 2015

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.
Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.
Authors: Dutcher S K
Published: September 2015

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.
Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.
Authors: Ashman C R , Davidson R L
Published: September 2015

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.
Transcriptional regulation of hemoglobin switching in chicken embryos.
Authors: Groudine M , Peretz M , Weintraub H
Published: September 2015

We used recombinant chicken deoxyribonucleic acid clones containing embryonic and adult beta-globin genes and "runoff" endogenous nuclear transcription to investigate the expression of embryonic and adult beta-globin genes during hematopoiesis in the developing chicken embryo. Purified, cloned deoxyribonucleic acids were digested with various restriction enzymes, separated on agarose gels, blotted to nitrocellulose, and annealed with purified nuclear [32P]ribonucleic acid synthesized in vitro from embryonic or adult red cell nuclei. Transcription of the respective globin genes was assayed by hybridization of nuclear [32P]ribonucleic acid to specific restriction fragments containing adult or embryonic coding sequences. Our results indicate that little, if any, transcription from the adult or embryonic beta-globin genes is detectable in the heterologous red cell nuclei, even under conditions in which ribonucleic acid processing probably does not occur.
Stage-specific synthesis of proteins complexed to ribonucleoprotein particles and ribosomes in zoospores of Blastocladiella emersonii.
Authors: Jaworski A J , Stumhofer P
Published: September 2015

In Blastocladiella emersonii zoospores, a set of proteins was found associated with the ribosomes and free ribonucleoprotein particles distinct from the ribosomes and polyribosomes. These proteins were designated P120, P105, P64, P56, and P42 based on their molecular weights determined by gel electrophoresis. Synthesis of these proteins was detected only during late sporulation just before the time polyadenylated ribonucleic acid accumulates in the sporangia. These proteins banded in isopycnic metrizamide gradients at densities of 1.31 and 1.27 g/cm3, which corresponded to the densities of the ribosomes and free ribonucleoprotein particles, respectively. Comparison of the distribution of the proteins in sucrose versus metrizamide gradients suggested that P105 was removed from the free ribonucleoprotein particles before complexing with the ribosomes. During germination, these proteins disappeared from the ribosomal fractions, with kinetics corresponding to the resumption of protein synthesis. Another protein (P178) was observed to bind to the ribosomes before the onset of protein synthesis during germination. Cycloheximide did not block the addition of this protein to the monoribosomes.
Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.
Authors: Gray M W
Published: September 2015

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.
Physical evidence for a Saccharomyces cerevisiae transposable element which carries the his4C gene.
Authors: de Bruijn F , Greer H
Published: September 2015

A Saccharomyces cerevisiae transposable element which carries the his4C structural gene and which is capable of transposition, excision, and mutator activity is described. Physical evidence is presented for transposition of the his4C deoxyribonucleic acid sequences to a new location in the genome and for precise excision of these transposed deoxyribonucleic acid sequences in spontaneous his4C- segregants.
Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.
Authors: Cooper J A , Hunter T
Published: September 2015

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.
Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.
Authors: Della Valle G , Fenton R G , Basilico C
Published: September 2015

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.
Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae.
Authors: Mizunaga T , Tkacz J S , Rodriguez L , Hackel R A , Lampen J O
Published: September 2015

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.
Isolation and characterization of the dopa decarboxylase gene of Drosophila melanogaster.
Authors: Hirsh J , Davidson N
Published: September 2015

We have isolated chromosomal deoxyribonucleic acid clones containing the Drosophila dopa decarboxylase gene. We describe an isolation procedure which can be applied to other nonabundantly expressed Drosophila genes. The dopa decarboxylase gene lies within or very near polytene chromosome band 37C1-2. The gene is interrupted by at least one intron, and the primary mode of regulation is pretranslational. At least two additional sequences hybridized by in vivo ribonucleic acid-derived probes are found within a 35-kilobase region surrounding the gene. The developmental profile of ribonucleic acid transcribed from one of these regions differs from that of the dopa decarboxylase transcript.
Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila.
Authors: Pratt K , Hattman S
Published: September 2015

Deoxyribonucleic acid (DNA) of the transcriptionally active macronucleus of Tetrahymena thermophila is methylated at the N6 position of adenine to produce methyladenine (MeAde); approximately 1 in every 125 adenine residues (0.8 mol%) is methylated. Transcriptionally inert micronuclear DNA is not methylated (< or = 0.01 mol% MeAde; M. A. Gorovsky, S. Hattman, and G. L. Pleger, J. Cell Biol. 56:697-701, 1973). There is no detectable cytosine methylation in macronuclei in Tetrahymena DNA (< or = 0.01 mol% 5-methylcytosine). MeAde-containing DNA sequences in macronuclei are preferentially digested by both staphylococcal nuclease and pancreatic deoxyribonuclease I. In contrast, there is no preferential release of MeAde during digestion of purified DNA. These results indicate that MeAde residues are predominantly located in "linker DNA" and perhaps have a function in transcription. Pulse-chase studies showed that labeled MeAde remains preferentially in linker DNA during subsequent rounds of DNA replication; i.e., there is little, if any, movement of nucleosomes during chromatin replication. This implies that nucleosomes may be phased with respect to DNA sequence.
Monocistronic transcription is the physiological mechanism of sea urchin embryonic histone gene expression.
Authors: Mauron A , Levy S , Childs G , Kedes L
Published: September 2015

We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.
Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.
Authors: Zakian V A , Wagner D W , Fangman W L
Published: September 2015

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.
Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.
Authors: Zimmermann W , Weissbach A
Published: September 2015

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.
Actin gene expression in developing sea urchin embryos.
Authors: Crain W R , Durica D S , Van Doren K
Published: September 2015

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.
Clonal analysis of the late stages of erythroleukemia induced by two distinct strains of Friend leukemia virus.
Authors: Mager D , MacDonald M E , Robson I B , Mak T W , Bernstein A
Published: September 2015

We observed striking differences between the tumorigenic colony-forming cells present in the spleens of mice late after infection with the anemia-inducing strain of Friend leukemia virus (strain FV-A) and those present after infection with the polycythemia-inducing strain (strain FV-P). Cells within primary colonies derived from FV-A- and FV-P-transformed cells (CFU-FV-A and CFU-FV-P, respectively) contained hemoglobin and spectrin, indicating that the CFU-FV-A and CFU-FV-P were transformed erythroid progenitor cells. The proportion of cells containing hemoglobin was relatively high (> 25%) in newly isolated cell lines derived from CFU-FV-P colonies, whereas cell lines derived from CFU-FV-A colonies had only low levels (0 to 2%) of hemoglobin-containing cells. A high proportion of the cell lines derived from CFU-FV-A colonies responded to pure erythropoietin and accumulated spectrin and hemoglobin, whereas the cell lines derived from CFU-FV-P colonies did not. A cytogenetic analysis indicated that primary CFU-FV-P colony cells were diploid, whereas chromosomal aberrations were observed in the immediate progeny of CFU-FV-A. The presence of unique chromosomal markers in the majority of the cells within individual colonies derived from CFU-FV-A suggested that these colonies originated from single cells. Finally, leukemic progenitor cells transformed by strain FV-A appeared to have an extensive capacity to self-renew (i.e., form secondary colonies in methylcellulose), whereas a significant proportion of the corresponding cells transformed by strain FV-P did not. In addition, the self-renewal capacity of both CFU-FV-A and CFU-FV-P increased as the disease progressed. From these observations, we propose a model for the multistage nature of Friend disease; this model involves clonal evolution and expansion from a differentiating population with limited proliferative capacity to a population with a high capacity for self-renewal and proliferation.
No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.
Authors: Oppenheim A , Horowitz A T
Published: September 2015

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.
Purification of vacuoles from Neurospora crassa.
Authors: Vaughn L E , Davis R H
Published: September 2015

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.
Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency on qa-1 function.
Authors: Reinert W R , Patel V B , Giles N H
Published: September 2015

An in vitro protein-synthesizing system (rabbit reticulocyte) was programmed with total polyadenylated messenger ribonucleic acid from wild type and various mutants in the qa gene cluster of Neurospora crassa. The products of two of the qa genes, quinate dehydrogenase (qa-3+) and dehydroshikimate dehydratase (qa-4+), were identified by specific immunoprecipitation and sodium dodecyl sulfate-slab gel electrophoresis. The results indicated that for both genes induction of a specific enzyme activity by quinic acid depends on the de novo synthesis of a specific polypeptide and on the de novo appearance of specific messenger ribonucleic acid detectable by the in vitro translation assay. Furthermore, the results indicated that the appearance of this messenger ribonucleic acid is under the control of the qa-1 gene. The simplest interpretation of these results appears to be that induction of enzyme activity in the qa system is mediated by events at the transcriptional level.
Control of initiation and elongation of cilia during ciliary regeneration in Tetrahymena.
Authors: Hadley G A , Williams N E
Published: September 2015

Tetrahymena thermophila strain B could regenerate approximately 10% of its somatic ciliary mass in concentrations of cycloheximide believed to block all cytoplasmic protein synthesis. A quantitative study of the relative numbers and lengths of cilia regenerated in the presence and absence of cycloheximide under a variety of conditions suggested that specific initiation and elongation protein factors are involved in the control of ciliary morphogenesis in Tetrahymena.
Synthesis of repressible acid phosphatase in Saccharomyces cerevisiae under conditions of enzyme instability.
Authors: Bostian K A , Lemire J M , Halvorson H O
Published: September 2015

The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.
Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.
Authors: Chan R K , Otte C A
Published: September 2015

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").
Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.
Authors: Oliver N A , Wallace D C
Published: September 2015

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria.
Measurement of transcribed human X-chromosomal DNA sequences transferred to rodent cells by chromosome-mediated gene transfer.
Authors: McBride O W , Olsen A S , Aulakh G S , Athwal R S
Published: September 2015

Transfer of genetic information can be effected by incubation of cultured eucaryotic cells with isolated metaphase chromosomes. In most cases, a resulting transformed cell contains only a fragment of a donor chromosome. The amount of transferred donor DNA has been quantified in 11 independent mouse A9 transformants by nucleic acid hybridization analysis. Each transformant had been selected for hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8) transfer and contained part of the human X chromosome. A labeled probe of transcribed human X-chromosomal DNA was prepared by hybridization of nick-translated unique-sequence human DNA with whole cellular RNA from a human-mouse hybrid cell line, A9/HRBC2-A, containing a single human chromosome., X. The amount of human X-chromosomal DNA in the transformants was quantitated by comparing the hybridization of this probe with transformant and A9/HRBC2-A DNAs. Two unstable transformants which had a microscopically detectable donor chromosome fragment contained 15% of the human X-chromosomal single-copy DNA. Four other unstable transformants contained 4 to 7% of human X-chromosomal DNA sequences. The transferred DNA was below the level of detection in three other unstable and in all three stable transformants. We conclude that the initial transfer event can introduce a substantial amount of genetic information but only smaller amounts of DNA are stably incorporated by integration.
Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes.
Authors: Gottlieb M , Dwyer D M
Published: September 2015

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.
Syntheses and stabilities of proteins related to the polyoma small T antigen in Escherichia coli.
Authors: Horwich A , Koop A H , Eckhart W
Published: September 2015

We compared the syntheses and turnovers of two proteins related to the polyoma small T antigen synthesized in Escherichia coli from plasmids containing polyoma genomic segments joined to lac control elements. A protein with an authentic polyoma N terminus was more unstable than a protein with N-terminal amino acids derived from beta-galactosidase. Both were more unstable than most bacterial proteins.
Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.
Authors: Gillies S , Stollar V
Published: September 2015

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.