bluuebunny/crossref_metadata_2025 huggingface dataset:
Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.
Authors: Simon A E , Taylor M W , Bradley W E , Thompson L H
Published: September 2015

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.
Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.
Authors: Shen Y M , Hirschhorn R R , Mercer W E , Surmacz E , Tsutsui Y , Soprano K J , Baserga R
Published: September 2015

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.
Murine cell complementation of a cold-sensitive defect for simian virus 40 replication in simian cells.
Authors: Fischer-Fantuzzi L , Vesco C
Published: September 2015

Mouse 3T3 fibroblasts were found to complement, in simian cell variants semipermissive to simian virus 40, a cold-sensitive defect of an early function, but not a nonconditional defect of viral uncoating. The variant simian cells could rescue simian virus 40 from 3T3 transformants, and this capacity was not temperature dependent.
In vivo catenation and decatenation of DNA.
Authors: Mertz J E , Miller T J
Published: September 2015

We have noted previously that when circular, but not linear, DNA or chromatin was injected into Xenopus laevis oocytes, much of it went through an intermediate form in which it did not readily enter an agarose gel; after a few hours, it reappeared as monomer DNA that had acquired its full complement of nucleosomes (T. J. Miller and J. E. Mertz, Mol. Cell. Biol. 2:1581-1593, 1982). We determined, using electron microscopy and a variety of biochemical techniques, the structure of this aggregated material. Most of it was oligomeric and multimeric catenanes of the injected sample. In addition, injection of DNA that had been catenated in vitro with DNA gyrase resulted in the conversion of most of it back to monomer circles. These findings demonstrate directly that both catenation and decatenation of DNA occur in vivo under physiological conditions. Whether these reactions play a crucial role in nucleosome formation, as well as in DNA replication and recombination, remains to be determined.
Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression.
Authors: Williamson V M , Cox D , Young E T , Russell D W , Smith M
Published: September 2015

Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene.
Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.
Authors: Murray M J , Kaufman R J , Latt S A , Weinberg R A
Published: September 2015

The transcriptional promoter of the Harvey sarcoma virus long terminal repeat has been used to construct a biologically active dihydrofolate reductase chimera. The construction placed the long terminal repeat at the 5' end of a dihydrofolate reductase cDNA. This chimera mediated methotrexate resistance when introduced into wild-type NIH3T3 mouse cells by transfection. The chimeric sequences were expressed in the form of polyadenylated RNA and dihydrofolate reductase protein and were amplified when the methotrexate-resistant transfectants were selected to grow in increasing methotrexate concentrations. This chimera was dominant acting and able to confer a methotrexate-resistant phenotype on wild-type NIH3T3 cells. It has been used in cotransfection experiments with DNA from human tumor cells to obtain foci of methotrexate-resistant transformed NIH3T3 cells resulting from uptake of exogenous DNA. The transfected methotrexate-resistant cells carried double minute chromosomes that appeared to contain DNA acquired during transfection.
Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.
Authors: Solari A , Deutscher M P
Published: September 2015

A survey of RNases in Xenopus laevis oocytes has been carried out to identify potential tRNA-processing enzymes in this system. Using a variety of specific and nonspecific substrates, we have shown that oocytes contain multiple RNases with various specificities. Three activities that could cleave the extraneous residues from the artificial tRNA precursor, tRNA-C-[14C]U-C, to generate a substrate for -C-C-A addition by tRNA nucleotidyltransferase were identified. One of these was a cytoplasmic exonuclease which generated predominantly tRNA-C, whereas the other two were nuclear endonucleases which cleaved the precursor to generate tRNA-N. The possible involvement of these activities in 3' tRNA processing in oocytes is discussed.
Hepatitis B virus encodes an RNA polymerase III transcript.
Authors: Standring D N , Rall L B , Laub O , Rutter W J
Published: September 2015

We demonstrated that cloned hepatitis B virus (HBV) DNA directs the synthesis of a 700-base RNA (HBV 700) by RNA polymerase III in a cell-free transcription system. HBV 700 is the only transcript known to originate from the viral short strand and has been mapped to the region between roughly 1,635 and 954 base pairs on the viral map, between the surface and core antigen coding sequences but overlapping and opposing the putative DNA polymerase and B protein genes. The in vitro initiation sites for the HBV 700 and core antigen RNAs are only 50 bases apart, suggesting that these two genes may be coordinately regulated. Moreover, both of these initiation sites appear to lie within the approximately 300-base double-stranded region (the nick region) between the 5' end of the HBV short strand and the nick in the viral long strand. We found two unusual sequence elements in the nick region that are conserved between the human and woodchuck viruses.
Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts.
Authors: Collins M L , Wu J S , Santiago C L , Hendrickson S L , Johnson L F
Published: September 2015

We have studied the metabolism of dihydrofolate reductase (DHFR) RNA in cells synchronized in the G1 phase of the cell cycle by starvation for isoleucine and glutamine. The relative content and stability of DHFR mRNA and the relative rate of transcription of the DHFR gene are similar in starved and exponentially growing cells. However, the relative rate of labeling of DHFR mRNA is about three times lower in starved cells than in exponentially growing cells. When the starved cells are stimulated to reenter the cell cycle by feeding them with complete medium, the relative rate of labeling of DHFR mRNA increases about fourfold within 6 h. However, the relative rate of transcription of the DHFR gene changes very little during this period. Continuous labeling experiments show that starved cells convert DHFR heterogeneous nuclear RNA into cytoplasmic DHFR mRNA much more slowly than serum-limited or exponentially growing cells. Pulse-chase experiments show that DHFR mRNA sequences contained in DHFR heterogeneous nuclear RNA appear to be conserved in starved cells. In addition, the content of DHFR RNA sequences in the nuclei of starved cells is about three times greater than that in exponentially growing cells. Delayed processing of DHFR heterogeneous nuclear RNA is also observed when exponentially growing cells are treated with inhibitors of protein synthesis. Our results suggest that, although delayed processing leads to a decrease in the initial labeling rate of DHFR mRNA, it does not result in a decrease in the actual rate of production of the message.
Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions.
Authors: Lo C W
Published: September 2015

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.
Organization and evolution of the actin gene family in sea urchins.
Authors: Johnson P J , Foran D R , Moore G P
Published: September 2015

Genomic libraries of the sea urchins Strongylocentrotus franciscanus and Lytechinus pictus were screened with an actin cDNA clone from Strongylocentrotus purpuratus. Four nonoverlapping clones were isolated and characterized from the S. franciscanus library; three were isolated and characterized from the L. pictus library. Linked genes having the same transcriptional orientation were found on all S. franciscanus clones. Three clones contained two actin genes each; the other clone contained three. In contrast, the L. pictus clones contained only one actin gene. Comparison of actin genomic clones from these three species indicated a difference in the genomic organization of sea urchin actin genes in that the genes appear to be more highly clustered in S. franciscanus than in S. purpuratus and L. pictus. Genomic dot blots and reassociation kinetics demonstrated that the copy number of actin genes in all three species is 15 to 20. Nucleotide sequence homology of actin genes within and among the species was measured by thermal elution. These experiments indicated that there is a high degree of interspecies actin gene sequence homology but that, within each species, actin gene sequences may differ by as much as 30%. Sequencing of two S. franciscanus actin genes revealed introns at the same amino acid positions, 121 and 204, reported for S. purpuratus actin genes. These data demonstrated that the genomic copy number, the transcriptional orientation of linked genes, and, to the extent studied, the intron position of actin genes have evolved similarly in these three species. In contrast, significant change has occurred in the chromosomal arrangement of sea urchin actin genes.
Empirical equation that can be used to determine genetic map distances from tetrad data.
Authors: Ma C , Mortimer R K
Published: September 2015

An empirical equation has been developed that can be used to calculate genetic map distances from tetrad data with good accuracy for distances of up to at least 120 centimorgans.
Regulation of histone mRNA production and stability in serum-stimulated mouse 3T6 fibroblasts.
Authors: DeLisle A J , Graves R A , Marzluff W F , Johnson L F
Published: September 2015

We measured the content and metabolism of histone mRNA in mouse 3T6 fibroblasts during a serum-induced transition from the resting to growing state. The content of several histone H3 and H2b mRNAs was measured by an S1 nuclease procedure. All of these increase in parallel by a factor of about 50 during S phase. However, the rate of H3 gene transcription increased only fivefold during this period, as determined in an in vitro transcription assay. This suggests that histone mRNA content is also controlled at the posttranscriptional level. When resting cells were serum stimulated in the presence of cytosine arabinoside, the rate of H3 gene transcription increased to about the same extent as that in control-stimulated cells. However, cytoplasmic H3 mRNA content increased only five to seven-fold. The half-life of H3 mRNA during S phase was about 4 to 5 h. When cytosine arabinoside was added to cells in the S phase, the half-life of the message decreased to about 15 min. The rapid turnover of H3 mRNA was prevented when the drug was added in the presence of cycloheximide or puromycin. The rate of H3 gene transcription decreased by only 35% after treatment with cytosine arabinoside. These results suggest that H3 gene transcription is not tightly coupled to DNA replication but is controlled temporally during the resting to growing transition. However, there is a correlation between the rate of DNA synthesis and the stability of histone H3 mRNA.
Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.
Authors: Kelly L J , Kelly R , Ennis H L
Published: September 2015

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.
Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.
Authors: Bhat R A , Metz B , Thimmappaya B
Published: September 2015

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.
Differential expression of porcine major histocompatibility DNA sequences introduced into mouse L cells.
Authors: Satz M L , Singer D S
Published: September 2015

The expression of a porcine genomic DNA segment containing a major histocompatibility gene and its chromatin structure in mouse L cells have been investigated. The transformed cells, which contain about two copies of the 17.8-kilobase pig DNA insert per haploid genome, stably and uniformly express major histocompatibility antigen on their surfaces. This expression is the result of differential transcription of the 3-kilobase major histocompatibility gene; the other 14 kilobases of pig sequences flanking the coding sequence are not transcribed. Although the entire pig DNA segment is packaged into nucleosomes, only the transcriptionally active DNA sequences are packaged in a DNase I-sensitive conformation. These results suggest that the expression of this foreign DNA is actively regulated in L cells.
Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.
Authors: Olsen A S , Triemer D F , Sanders M M
Published: September 2015

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.
Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection.
Authors: Kao H T , Nevins J R
Published: September 2015

A cDNA copy of the major human heat shock mRNA was cloned. The clone is complementary to the mRNA encoding the major 70-kilodalton heat shock protein as shown by hybrid arrest translation. We utilized the cloned DNA to measure induction of the gene during adenovirus infection. The mRNA increases in abundance approximately 100-fold during a wild-type adenovirus infection but does not increase more than 2-fold during an infection in which there is no E1A gene function [high multiplicity of infection of an E1A (-) mutant]. Furthermore, by measuring transcription in isolated nuclei, we found that the induction was transcriptional and was mediated by the E1A gene product. The induction was not maintained, however. After a peak level was obtained, transcription returned to preinfection levels. This decline was also reflected in the cytoplasmic mRNA abundance indicating a rapid turnover of the heat shock mRNA. This rapid turnover of the heat shock mRNA appears to be induced by the viral infection since the heat shock mRNA was found to be stable when synthesized in an adenovirus-transformed cell line.
Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.
Authors: Wahl G M , Vitto L , Rubnitz J
Published: September 2015

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.
Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.
Authors: Ardeshir F , Giulotto E , Zieg J , Brison O , Liao W S , Stark G R
Published: September 2015

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.
U1 small nuclear RNA genes are located on human chromosome 1 and are expressed in mouse-human hybrid cells.
Authors: Lund E , Bostock C , Robertson M , Christie S , Mitchen J L , Dahlberg J E
Published: September 2015

The majority, and perhaps all, of the genes for human U1 small nuclear RNA (U1 RNA) were shown to be located on the short arm of human chromosome 1. These genes were mapped by Southern blot analysis of DNA from rodent-human somatic cell hybrids, using the 5' region of a human U1 RNA gene as a human-specific probe. This probe hybridized to DNA fragments present only in digests of total human DNA or to the DNAs of cell lines which contained human chromosome 1. The major families of human U1 RNA genes were identified, but some human genes may have gone undetected. Also, the presence of a few U1 RNA genes on human chromosome 19 could not be ruled out. In spite of the lack of extensive 5'-flanking-region homology between the human and mouse U1 RNA genes, the genes of both species were efficiently transcribed in the hybrid cells, and the U1 RNAs of both species were incorporated into specific ribonucleoprotein particles.
Presence of a Kirsten murine sarcoma virus ras oncogene in cells transformed by 3-methylcholanthrene.
Authors: Parada L F , Weinberg R A
Published: September 2015

Oncogenes have previously been reported in the DNAs of mouse fibroblast lines which had become transformed after in vitro exposure to the carcinogen 3-methylcholanthrene. These oncogenes are now shown to be versions of the cellular Kirsten ras gene and are therefore homologous to oncogenes detected in a variety of human tumor DNAs.
Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.
Authors: Firestone G L , Yamamoto K R
Published: September 2015

We have isolated mutant derivatives of M1.54 (a mammary tumor virus [MTV]-infected rat hepatoma [HTC] cell line containing multiple integrated proviruses) that fail to express hormone-inducible cell surface viral glycoproteins. In wild-type M1.54, the synthetic glucocorticoid dexamethasone selectively stimulates the rate of synthesis of MTV RNA. In addition, dexamethasone is essential for posttranslational maturation of three of the four cell surface viral glycoproteins processed from the MTV glycosylated precursor polyprotein; the fourth mature species is produced constitutively. Two mutant phenotypes are described; each contains glucocorticoid receptors that are indistinguishable from the wild-type receptor with respect to hormone affinity, intracellular concentration, nuclear translocation efficiency, DNA-cellulose chromatography, and sedimentation rate. In one class, represented by the mutant line CR1, dexamethasone fails to stimulate the low basal rate of MTV gene transcription; surprisingly, hormonal regulation of tyrosine aminotransferase activity is also defective in CR1, whereas several other cellular responses to dexamethasone are normal. In the second class of mutants, represented by CR4, dexamethasone stimulates synthesis of MTV transcripts indistinguishable from those produced in M1.54, but only the constitutive cell surface viral glycoprotein is expressed. Thus, these mutants define two distinct and novel aspects of glucocorticoid regulated gene expression in HTC cells: CR4 contains a defect in a hormone inducible protein maturation pathway that acts on specific viral (and presumably cellular) precursor polypeptides, whereas the lesion in CR1 appears to affect the expression of a subset of the gene products normally under glucocorticoid control in M1.54.
Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.
Authors: Mayrand S , Pederson T
Published: September 2015

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S.A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25 degrees C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25 degrees C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations.
Excision and replication of extrachromosomal DNA of pea (Pisum sativum).
Authors: Van't Hof J , Bjerknes C A , Delihas N C
Published: September 2015

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.
Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.
Authors: van Daalen Wetters T , Coffino P
Published: September 2015

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.
Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene.
Authors: Cole C N , Santangelo G M
Published: September 2015

Bal31 nuclease was used to resect the herpes simplex virus type 1 thymidine kinase (tk) gene from its 3' end, and a plasmid, pTK206, was isolated that lacked the processing and polyadenylation signals normally found at the 3' end of the gene. The wild-type gene, pTK2, and pTK206 were each transferred to pSV010, a plasmid containing the simian virus 40 (SV40) origin of DNA replication, allowing replication and analysis of the patterns of transcription in Cos-1 cells. Fragments of DNA containing processing and polyadenylation signals from SV40 and polyoma virus were inserted into the 3' end of the resected tk gene, pTK206. We found that tk gene expression requires a processing and polyadenylation signal, that signals from SV40 and polyoma virus could substitute for the herpes simplex virus tk signal, and that considerable differences in the levels of tk mRNA were present in Cos-1 cells transfected by these gene constructs. In addition, tk gene expression was restored to a low level after the insertion of an 88-base-pair fragment from the middle of the SV40 early region. Processing and polyadenylation do not occur in the vicinity of this fragment in SV40, even though it contains the hexanucleotide 5'-AAUAAA-3'.
Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.
Authors: Andrulis I L , Duff C , Evans-Blackler S , Worton R , Siminovitch L
Published: September 2015

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.
DNA sequence of two linked actin genes of sea urchin.
Authors: Schuler M A , McOsker P , Keller E B
Published: September 2015

DNA sequences have been determined for two actin genes which are closely linked in the genome of the sea urchin Strongylocentrotus purpuratus. The two genes have the same 5'-3' orientation; they were apparently formed originally by tandem gene duplication. The amino acids encoded by the two genes closely resemble those of cytoplasmic actins of mammals and slime molds and differ somewhat from those of mammalian muscle actin. Actin gene 1 had been tentatively identified earlier as the gene for an embryonic cytoplasmic actin by the homology of the 3' noncoding region with that of the cDNA of an embryonic actin mRNA from S. purpuratus. The DNA sequence of gene 1 shows presumptive signals for the initiation and termination of transcription which would govern the formation of a mature mRNA of 1.9 kilobases. Both actin genes 1 and 2 have introns in their coding regions at codons 121/122 and 204. These positions for actin introns have been reported so far only in the rat, not in lower organisms. The divergence of the sequences of these coding-region introns in the two actin genes is 66%, suggesting that the genes diverged about 90 million years ago. By contrast to the introns, the coding regions have been highly conserved; the amino acids of the two genes differ by only 1.3%, and the silent sites of the codons differ by only 12%.
Increased phosphorylation of vinculin on tyrosine does not occur during the release of stress fibers before mitosis in normal cells.
Authors: Rosok M J , Rohrschneider L R
Published: September 2015

We examined the extent of phosphorylation of vinculin on tyrosine after pulse labeling synchronized cells with 32PO4 at various stages of the cell cycle. No significant increase was observed in the incorporation of radioactivity into vinculin phosphotyrosine throughout the cell cycle, suggesting that the increased rate of phosphorylation of vinculin on tyrosine may not be the major event regulating stress fiber dissolution before mitosis.
Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.
Authors: Thill G P , Kramer R A , Turner K J , Bostian K A
Published: September 2015

The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.
Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.
Authors: Oishi K K , Tewari K K
Published: September 2015

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20% of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in SalI-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 +/- 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.
Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.
Authors: Wickner R B
Published: September 2015

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.
Multiple, Tandem Plasmid Integration inSaccharomyces cerevisiae
Authors: Orr-Weaver Terry L. , Szostak Jack W.
Published: September 2015

Nonreplicating plasmids transformSaccharomyces cerevisiaeby recombining with a homologous site in the genome. Frequently, multiple copies of the plasmid integrate in a tandem array. We show that, after transformation with restriction enzyme-cut plasmids, most, if not all, multimers arise by sequential integration of plasmid molecules into the same genomic location.