bluuebunny/crossref_metadata_2025 huggingface dataset:
Abelson murine leukemia virus-transformed cells that lack p53 protein synthesis express aberrant p53 mRNA species.
Authors: Wolf D , Admon S , Oren M , Rotter V
Published: September 2015

Cells of the Abelson murine leukemia virus-transformed line L12 that lack the p53 protein also lack polyadenylated mRNA capable of directing the synthesis of p53 in a cell-free system. Direct analysis of stable polyadenylated mRNA from a variety of cell lines shows that all p53 producers shared a common mRNA species (2.0 kilobases) which hybridized with a p53-specific cDNA probe. This species, which appears to be the mature, normal-sized p53 mRNA, was totally undetectable in L12 cells, which did not produce p53 in vivo. However, L12 cells contained two major p53-specific mRNA species of a substantially larger size (3.5 and 6.5 kilobases) than the p53-specific mRNA in the p53-producing cells. Genomic DNA analysis uncovered an apparent alteration in the 5' proximal part of only one p53 gene, which is unique to the L12 cell line. It is thus possible that the nonproducer phenotype of L12 cells is due at least in part to an alteration within a p53-specific DNA sequence. These findings define a system in which production of p53 appears to be efficiently regulated at the level of stable mRNA and which can be used to study the mechanisms controlling p53 expression in Abelson murine leukemia virus-transformed cells.
Relationship of DNA methylation level to the presence of heterochromatin in mealybugs.
Authors: Scarbrough K , Hattman S , Nur U
Published: September 2015

Purified nuclear DNA from two mealybug species was analyzed for its 5-methylcytosine (m5C) content by reversed-phase high-pressure liquid chromatography. We observed that the percent m5C (percentage of cytosines which are methylated) varied between the two species, between males and females of the same species, and between lines with and without supernumerary B chromosomes. This is the first case of a sex-specific difference in overall DNA methylation level. In contrast to a recent report (Deobagkar et al., J. Biosci. [India] 4:513-526, 1982), we found no other modified bases in the DNA. Overall, the percent m5C in Pseudococcus obscurus was two to three times higher than in Pseudococcus calceolariae. In both species, the percent m5C in males was higher than in females, although only in P. calceolariae was the difference statistically significant (0.68 +/- 0.02 versus 0.44 +/- 0.04). The high m5C content in males was correlated with the presence of a paternally derived, genetically inactive set of chromosomes which is facultatively heterochromatic. The presence of constitutive heterochromatin, however, was associated with a lower m5C content. Thus, for example, the percent m5C in females of a P. obscurus line with heterochromatic B chromosomes (1.09 +/- 0.04) was significantly lower than that of a related line lacking such chromosomes (1.26 +/- 0.06). Our findings are discussed with respect to the possible relationship between DNA methylation and heterochromatization.
Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.
Authors: Roeder G S , Smith M , Lambie E J
Published: September 2015

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.
Expression of complete chicken thymidine kinase gene inserted in a retrovirus vector.
Authors: Bandyopadhyay P K , Temin H M
Published: September 2015

The chicken thymidine kinase (tk) gene was inserted into spleen necrosis virus. Thymidine kinase activity was expressed even when the promoter and terminator sequences for tk RNA synthesis were retained. When the promoter was present in the same orientation as the promoter in the long terminal repeat of the virus, deletions occurred both in the virus and in the tk gene, and the thymidine kinase-transforming activity of the recovered virus was low. Splicing of apparent intervening sequences in the tk gene was also observed. When the orientation of the tk promoter was opposite to the promoter in the long terminal repeat, virus synthesis was diminished, whereas thymidine kinase activity was expressed at an elevated level compared with virus in which the promoter was in the same orientation. However, when the apparent tk promoter was deleted from virus with the tk gene in the opposite orientation, a high level of virus synthesis was observed, probably as a result of absence of interference of RNA synthesis from converging promoters. The intervening sequences in the virus in which the promoters were in opposite orientation were not spliced.
Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.
Authors: Imperiale M J , Kao H T , Feldman L T , Nevins J R , Strickland S
Published: September 2015

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.
Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation.
Authors: Imperiale M J , Nevins J R
Published: September 2015

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.
DNA methylation and expression of HLA-DR alpha.
Authors: Reitz M S , Mann D L , Eiden M , Trainor C D , Clarke M F
Published: September 2015

B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DR alpha DNA sequences detected by a cloned DR alpha cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DR alpha mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DR alpha mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DR alpha mRNA, and the partial demethylation of the DR alpha DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DR alpha mRNA; the HpaII sites within the BglII fragment of DR alpha DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DR alpha locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DR alpha locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.
Frequent hereditable shutdown of murine retrovirus gene expression in murine cell lines.
Authors: Bestwick R K , Machida C A , Polonoff E , Kabat D
Published: September 2015

Friend spleen focus-forming virus shuts down its gene expression frequently (ca. 10(-3) per generation) in a cis-dominant hereditable fashion in various murine cells but much less frequently in rat cells (less than 10(-6) per generation). Thus, nonexpresser variants were isolated at high frequency from murine cell lines by immunoselection directed against virus-encoded cell surface glycoproteins and also simply by subcloning cells from lines which had been cultured for many generations. Studies of independently infected cell clones indicate that shutdown is a property of the cell line rather than of the specific proviral site. Nucleic acid blot analyses suggest that shutdown correlates with decreased transcription. Moreover, preliminary evidence indicates that other murine retroviruses also shut down frequently in murine but not in rat cells and that shutdown of replication-competent murine leukemia viruses with accompanying loss in interference to superinfection may be the rate-limiting reaction enabling cells to acquire multiple proviruses in their chromosomes. High-frequency shutdown in vivo would have important pathological consequences.
Three different macronuclear DNAs in Oxytricha fallax share a common sequence block.
Authors: Cartinhour S W , Herrick G A
Published: September 2015

The three members of a cross-hybridizing family of macronuclear DNAs (4,890, 2,780, and 1,640 base pairs) from the protozoan Oxytricha fallax have in common a conserved sequence block 1,300 to 1,550 base pairs long. Adjacent to the common block in the two larger DNAs are sequences which are unique to them, whereas the smallest DNA contains few if any additional sequences. The family reappears when the macronucleus is replaced after conjugation and can be detected in another O. fallax subspecies. In a random collection of cloned macronuclear DNAs, 6 of 15 hybridize to macronuclear DNA families. This high frequency suggests that families sharing common sequence blocks have an important role in macronuclear function.
Fine mapping of an immunoglobulin gene activator.
Authors: Queen C , Stafford J
Published: September 2015

It has recently been shown that the promoter of a kappa immunoglobulin gene is activated for transcription by a downstream sequence element. Here we mapped the activating element to a resolution of about 20 base pairs by constructing a series of deletions in the cloned kappa gene. After transfection of each deleted gene into myeloma cells, a transient expression assay was used to measure the level of transcription from the kappa promoter. We found that the activating element extends through about 200 base pairs and encompasses a region of sequence that is conserved between mouse and human genes. As successively deeper deletions were made into the conserved region from either the 5' or 3' side, the activating ability was lost gradually rather than abruptly. Although several short segments in this region are homologous to sequences in viral enhancers, they did not seem to play a dominant role in the activating effect. We also found that the activating element remained functional when reversed in orientation or when moved upstream of the kappa gene.
Structure of a human smooth muscle actin gene (aortic type) with a unique intron site.
Authors: Ueyama H , Hamada H , Battula N , Kakunaga T
Published: September 2015

A recombinant phage containing an actin gene (lambda Ha201) was isolated from a human DNA library and the structure of the actin gene was determined. The amino acid sequences deduced from the nucleotide sequences of lambda Ha201 were compared with those of six actin isoforms; they matched those of bovine aortic smooth muscle actin, except for codon 309, which was valine (GTC) in lambda Ha201 and alanine (GCN) in bovine aortic smooth muscle actin. Southern blot hybridization experiments showed that the gene of normal human cells did not have the TaqI-sensitive site around position 309, whereas half of the genes of HUT14 cells did. These results indicate that one allele of the aortic smooth muscle actin gene in HUT14 cells has a transition point mutation (C----T) at codon 309 and that the amino acid sequences of normal human aorta and bovine smooth muscle actins are probably identical. In addition to the five introns interrupting exons at codons 150, 204, and 267, and between codons 41 and 42 and 327 and 328, which are common to skeletal muscle and cardiac muscle actin genes, the smooth muscle actin gene has two more intron sites between codons 84 and 85 and 121 and 122. The previously unreported intron site between codons 84 and 85 is unique to the smooth muscle actin gene. The intron site between codons 121 and 122 is common to beta-actin genes but is not found in other muscle actin genes. A hypothesis is proposed for the evolutionary pathway of the actin gene family.
Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.
Authors: McClung J K , Kletzien R F
Published: September 2015

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.
Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.
Authors: Steinberg R A
Published: September 2015

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.
Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.
Authors: Brunke K J , Anthony J G , Sternberg E J , Weeks D P
Published: September 2015

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.
Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP.
Authors: Schulman H
Published: September 2015

Comparison of cyclic AMP- and calcium-dependent phosphorylation in rat brain cytosol reveals MAP-2 to be a common endogenous substrate. Examination of limited protease digestion patterns indicates that the two kinases phosphorylate MAP-2 at distinct sites and suggests that such phosphorylation may have differential effects on MAP-2 function.
Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.
Authors: Zitomer R S , Walthall D A , Rymond B C , Hollenberg C P
Published: September 2015

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.
Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.
Authors: Pekkala D , Heath B , Silver J C
Published: September 2015

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.
Effects of Ty insertions on HIS4 transcription in Saccharomyces cerevisiae.
Authors: Silverman S J , Fink G R
Published: September 2015

Insertion of two different Ty elements into the Saccharomyces cerevisiae HIS4 regulatory region eliminates transcription of HIS4. Transcription can be restored by genetic rearrangements involving the Ty element inserted at HIS4. Several deletions, an inversion, a translocation, and a gene conversion are capable of restoring HIS4 transcription. Some of the rearrangements result in new transcriptional initiation sites. One type of revertant of his4-912 results from recombination between the delta elements flanking the Ty element, leaving a solo delta in place of the complete Ty. Strains carrying a Ty912 delta at HIS4 are His- at 23 degrees C. Unlinked suppressors (SPT) lead to suppression of this His- phenotype and increase levels of the normal HIS4 transcript. These suppressor genes affect not only the amount of transcription from the normal HIS4 initiation site, but also that from new initiation sites within Ty sequences adjacent to HIS4.
The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.
Authors: Miesfeld R , Sollner-Webb B , Croce C , Arnheim N
Published: September 2015

The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells.
Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.
Authors: Ghosh P C , Wellner R B , Wu H C
Published: September 2015

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.
Detection of deletion mutations in pSV2gpt-transformed cells.
Authors: Tindall K R , Stankowski L F , Machanoff R , Hsie A W
Published: September 2015

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.
Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.
Authors: Johnston M , Davis R W
Published: September 2015

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.
Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.
Authors: Ariga H
Published: September 2015

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.
Stress mRNA metabolism in canavanine-treated chicken embryo cells.
Authors: White C N , Hightower L E
Published: September 2015

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.
UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells.
Authors: Maltzman W , Czyzyk L
Published: September 2015

Elevated levels of the p53 cellular tumor antigen have been previously observed in proliferating and transformed mammalian cells. We found that nontransformed mouse cells treated with either UV light or a UV-mimetic chemical carcinogen exhibited a rapid increase in the amount of p53. This stimulation can be explained, at least in part, on the basis of a post-translational stabilization of p53 which is independent of replicative DNA synthesis, consistent with p53 not being an adventitious product of proliferating cells. The results presented here are interpreted in light of the general hypothesis that p53 is involved in the preparation of mammalian cells for DNA synthesis.
Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.
Authors: Green L L , Dove W F
Published: September 2015

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.
Amphotropic retrovirus vector system for human cell gene transfer.
Authors: Sorge J , Wright D , Erdman V D , Cutting A E
Published: September 2015

Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus. This problem has been circumvented by segregating the viral genome into cis- and trans-acting components. By establishing helper cell lines that produce the trans-acting viral gene products, one can propagate the cis-acting component in them and harvest defective viral particles that contain only the cis-acting component. The cis-acting component can provide a useful vehicle for the highly efficient transfer of genes into target cells. The defective vector systems described to date, however, are restricted in host range to murine, avian, rat, and dog cells. We describe a helper-free vector system based entirely on an amphotropic murine virus with a wide mammalian host range, including the ability to carry out efficient gene transfer into human cells. We also describe a double mutation constructed in the trans-acting genome which reduces the frequency of replication-competent recombinant viruses to undetectable levels.
tk Enzyme expression in differentiating muscle cells is regulated through an internal segment of the cellular tk gene.
Authors: Merrill G F , Hauschka S D , McKnight S L
Published: September 2015

Thymidine kinase (tk) enzyme expression is shut down when cultured skeletal muscle cells terminally differentiate. This regulation is mediated by a rapid and specific decline in the abundance of cellular tk mRNA. tk-deficient mouse myoblasts were transformed to the tk-positive phenotype by using both the cellular tk gene of the chicken and the herpesvirus tk gene. Myoblasts transformed with the cellular tk gene effectively regulate tk enzyme activity upon terminal differentiation. Conversely, myoblasts transformed with the herpesvirus tk gene continue to express tk enzyme activity in postreplicative muscle cells. A regulated pattern of expression is retained when the promoter of the cellular tk gene is replaced by the promoter of the herpesvirus tk gene. Moreover, the cellular tk gene is appropriately regulated during terminal muscle differentiation when its 3' terminus is removed and replaced by the terminus of the viral tk gene. Thus, the element of the cellular tk gene sufficient to specify its regulation is entirely intragenic.
70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences.
Authors: Kothary R K , Jones D , Candido E P
Published: September 2015

RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.
Differentiation of two mouse cell lines is associated with hypomethylation of their genomes.
Authors: Bestor T H , Hellewell S B , Ingram V M
Published: September 2015

Methyl-accepting assays and a sensitive method for labeling specific CpG sites have been used to show that the DNA of F9 embryonal carcinoma cells decreases in 5-methylcytosine content by ca. 9% during retinoic acid-induced differentiation, whereas the DNA of dimethyl sulfoxide-induced Friend murine erythroleukemia (MEL) cells loses ca. 3.8% of its methyl groups. These values correspond to the demethylation of 2.2 X 10(6) and 0.9 X 10(6) 5'-CpG-3' sites per haploid genome in differentiating F9 and MEL cells, respectively. Fluorography of DNA restriction fragments methylated in vitro and displayed on agarose gels showed that demethylation occurred throughout the genome. In uninduced F9 cells, the sequence TCGA tended to be more heavily methylated than did the sequence CCGG, whereas this tendency was reversed in MEL cells. The kinetics of in vitro DNA methylation reactions catalyzed by MEL cell DNA methyltransferase showed that substantial numbers of hemimethylated sites accumulate in the DNA of terminally differentiating F9 and MEL cells, implying that a partial loss of DNA-methylating activity may accompany terminal differentiation in these two cell types.
Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.
Authors: Campisi J , Pardee A B
Published: September 2015

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.
Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation.
Authors: Hendricks M , Weintraub H
Published: September 2015

We have found that cytoskeletal extracts of cultured chicken embryo fibroblasts contain at least seven distinct polypeptides (two major and five minor) which cross-react with antiserum to chicken smooth muscle tropomyosin. These polypeptides range in apparent molecular weight from 31,000 to 47,000, and each is encoded by mRNAs which specifically hybridize to cloned muscle tropomyosin cDNAs. These nonmuscle tropomyosin species and their respective mRNAs are electrophoretically distinct from those of chicken skeletal muscle and appear by genomic DNA blotting to comprise a part of a multigene tropomyosin family. In Rous sarcoma virus-transformed chicken embryo fibroblasts, synthesis of the tropomyosins is differentially repressed such that the synthesis of the major species (cp35 and cp33, cytoskeletal proteins of molecular weight 35,000 and 33,000, respectively) and three minor species is drastically reduced, whereas the synthesis of two of the minor species (cp32 and cp31) remains essentially unchanged. Analysis of cellular mRNA and runoff nuclear transcription experiments indicate that the repression of tropomyosin synthesis by Rous sarcoma virus transformation occurs at the level of transcription. This repression of tropomyosin synthesis is partially mimicked in normal chicken embryo fibroblasts during incubation in high-NaCl medium, a condition in which chicken embryo fibroblasts acquire many characteristics of transformed cells.
Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels.
Authors: Focht R J , Adams S L
Published: September 2015

We analyzed the control of type I collagen synthesis in four kinds of differentiated cells from chicken embryos which synthesize very different amounts of the protein. Tendon, skin, and smooth muscle cells were found to have identical amounts of type I collagen RNAs; however, the RNAs had inherently different translatabilities, which were observed both in vivo and in vitro. Chondrocytes also had substantial amounts of type I collagen RNAs, even though they directed no detectable synthesis of the protein either in vivo or in vitro. Type I collagen RNAs in chondrocytes display altered electrophoretic mobilities, suggesting that in these cells the reduction in translational efficiency may be mediated in part by changes in the RNA structure. These data indicate that control of type I collagen gene expression is a complex process which is exerted at both transcriptional and post-transcriptional levels.
Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.
Authors: Cirullo R E , Wasmuth J J
Published: September 2015

Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.
Intussusception in gestational choriocarcinoma (not histologically proven), resolving spontaneously with chemotherapy
Authors: Ramessur Anisha , Openshaw Mark , Sarwar Naveed
Published: September 2015

We present a rare case of advanced gestational choriocarcinoma with small bowel metastatic involvement and intussusception, which presented acutely as a lower gastrointestinal bleed with symptomatic anaemia and haemoglobin 3.8 g/dL in a young woman. A diagnosis of gestational choriocarcinoma was made without biopsy, using a combination of clinical history, isolated elevated human chorionic gonadotropin markers of 77 000 IU/mL and radiological findings. Surgical intervention was too high risk due to the presence of active bleeding and increased vascularity surrounding the intussusception. Owing to the highly responsive nature of gestational choriocarcinoma to chemotherapy, frontline chemotherapy alone was used to reduce the size of the metastatic small bowel deposits, with subsequent resolution of the bleeding and intussusception. This is the first time chemotherapy alone has been used to successfully resolve small bowel intussusception secondary to metastatic choriocarcinoma that has been documented according to PubMed searches.
ST segment elevation myocardial infarction of a rare aetiology: an unexpected diagnosis
Authors: Mandal Kaushik , Karki Apurwa , Mangla Aditya
Published: September 2015

A 43-year-old man presenting with acute coronary syndrome with ST segment elevation myocardial infarction underwent urgent coronary angiography. During the intervention, the patient was found to have several multiple filling defects with dynamic obstruction in the left coronary circulation. Thrombectomy was performed on distal left anterior descending artery and 2nd diagonal artery lesions with balloon angioplasty, which was unsuccessful. Considering the dynamic obstruction in the angiogram, immediate imaging was performed for structural evaluation of the heart. Cardiac CT revealed a circumferential groove on the heart, suggesting an external compression leading to dynamic obstruction of the coronary arteries on angiogram. Cardiac hernia, a very rare aetiology, was suspected to be the culprit for the ST segment elevation myocardial infarction. Thoracoscopy was performed, which revealed congenital cardiac hernia and a fibrous pericardial band encircling the apex. A left mini thoracotomy was performed to release the constriction imposed over the heart, with improvement in circulation.