bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and characterization of the STE7 and STE11 genes of Saccharomyces cerevisiae.
Authors: Chaleff D T , Tatchell K
Published: September 2015

In the yeast Saccharomyces cerevisiae, haploid cells occur in one of the two cell types, a or alpha. The allele present at the mating type (MAT) locus plays a prominent role in the control of cell type expression. An important consequence of the elaboration of cell type is the ability of cells of one mating type to conjugate with cells of the opposite mating type, resulting in yet a third cell type, an a/alpha diploid. Numerous genes that are involved in the expression of cell type and the conjugation process have been identified by standard genetic techniques. Molecular analysis has shown that expression of several of these genes is subject to control on the transcriptional level by the MAT locus. Two genes, STE7 and STE11, are required for mating in both haploid cell types; ste7 and ste11 mutants are sterile. We report here the molecular cloning of STE7 and STE11 genes and show that expression of these genes is not regulated transcriptionally by the MAT locus. We also have genetically mapped the STE11 gene to chromosome XII, 40 centimorgans from ura4.
Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.
Authors: Greenspan D S , Weissman S M
Published: September 2015

The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.
Transcriptional regulation of a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response.
Authors: Berry-Lowe S L , Meagher R B
Published: September 2015

The effects of white light, far-red light, and darkness on the transcription of a soybean ribulose-1,5-biphosphate carboxylase small subunit gene, SRS1, were investigated. RNA was labeled with [alpha-32P]UTP in nuclei isolated from plants grown under different conditions of light and darkness and used to probe Southern blots and dot blots. The levels of small subunit mRNA synthesis were normalized to ribosomal RNA synthesis. We demonstrate that the SRS1 gene is transcribed at a rate 16- to 32-fold higher in plants grown in the light than in those grown in darkness. Transcription of the small subunit increased dramatically when plants grown in darkness were given 30 min to 6 h of light and then leveled off after 24 to 48 h of exposure. When light-grown seedlings were exposed to greater than 2 h of darkness, a gradual decrease in transcription was detected. This decrease in transcription reached basal dark-grown levels after 48 h of exposure to darkness. The increase in transcription in etiolated seedlings treated with white light for 15 min could be reduced to basal levels if the treatment was followed by treatment with far-red light for 15 min. In addition, transcription in ligh-grown seedlings was reduced to basal levels when plants were exposed to far-red light for 15 min. The transcription of this ribulose-1,5-biphosphate carboxylase small subunit gene is strongly positively regulated by white light, is negatively regulated by far-red light, and exhibits a classic phytochrome-linked response.
Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4.
Authors: DeLuca N A , Schaffer P A
Published: September 2015

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.
Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.
Authors: Stillman B W , Gluzman Y
Published: September 2015

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.
Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.
Authors: Cole C N , Stacy T P
Published: September 2015

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SV010 and pTK209R/SV010). Removal of an additional 7 bp (pTK312R/SV010) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SV010); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G + T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.
Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.
Authors: Lakshmikumaran M S , D'Ambrosio E , Laimins L A , Lin D T , Furano A V
Published: September 2015

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.
Chromosomal position and specific demethylation in enhancer sequences of germ line-transmitted retroviral genomes during mouse development.
Authors: Jähner D , Jaenisch R
Published: September 2015

The methylation pattern of the germ line-transmitted Moloney leukemia proviral genome was analyzed in DNA of sperm, of day-12 and day-17 embryos, and of adult mice from six different Mov substrains. At day 12 of gestation, all 50 testable CpG sites in the individual viral genomes as well as sites in flanking host sequences were highly methylated. Some sites were unmethylated in sperm, indicating de novo methylation of unique DNA sequences during normal mouse development. At subsequent stages of development, specific CpG sites which were localized exclusively in the 5' and 3' enhancer regions of the long terminal repeat became progressively demethylated in all six proviruses. The extent of enhancer demethylation, however, was tissue specific and strongly affected by the chromosomal position of the respective proviral genome. This position-dependent demethylation of enhancer sequences was not accompanied by a similar change within the flanking host sequences, which remained virtually unchanged. Our results indicate that viral enhancer sequences, but not other sequences in the M-MuLV genome, may have an intrinsic ability to interact with cellular proteins, which can perturb the interaction of the methylase with DNA. Demethylation of enhancer sequences is not sufficient for gene expression but may be a necessary event which enables the enhancer to respond to developmental signals which ultimately lead to gene activation.
Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.
Authors: Mathison L , Culbertson M R
Published: September 2015

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.
Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.
Authors: Chakrabarti S , Joffe S , Seidman M M
Published: September 2015

Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.
General method for cloning Neurospora crassa nuclear genes by complementation of mutants.
Authors: Akins R A , Lambowitz A M
Published: September 2015

We have developed a sib selection procedure for cloning Neurospora crassa nuclear genes by complementation of mutants. This procedure takes advantage of a modified N. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of DNA with recombinant plasmids containing the N. crassa qa-2+ gene. Here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. The identities of the putative clones were confirmed by mapping their chromosomal locations in standard genetic crosses and using restriction site polymorphisms as genetic markers. Because we can obtain very high N. crassa transformation frequencies, cloning can be accomplished with as few as five subdivisions of an N. crassa genomic library. The sib selection procedure should, for the first time, permit the cloning of any gene corresponding to an N. crassa mutant for which an appropriate selection can be devised. Analogous procedures may be applicable to other filamentous fungi before the development of operational shuttle vectors.
The spliced leader sequence of Trypanosoma brucei has a potential role as a cap donor structure.
Authors: Lenardo M J , Dorfman D M , Donelson J E
Published: September 2015

Trypanosoma brucei brucei and other trypanosomatid species are unique among eucaryotes because transcription of their protein-coding genes is discontinuous. The 5' ends of their mRNAs consist of an identical 35-nucleotide spliced leader which is encoded at a separate locus from that for the body of the protein-coding transcript. We show here that the spliced leader transcript contains a 5' cap structure and suggest that at least one function of the spliced leader sequence is to provide a cap structure to trypanosome mRNAs.
Multiple C4/Slp genes distinguished by expression after transfection.
Authors: Robins D M , Malissen M , Hood L , Ferreira A , Walthall D , Mitchell M
Published: September 2015

The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes.
Expression of the intracisternal A-particle is elevated during differentiation of embryonal carcinoma cells.
Authors: Howe C C , Overton G C
Published: September 2015

Three cDNA clones coding for the 3' region of the intracisternal A-particle (IAP), a mouse endogenous retrovirus, were isolated during screening of a library for genes whose expression was modulated during the retinoic acid-induced differentiation of the embryonal carcinoma cell line F9 into parietal endoderm-like (PE-like) cells. In contrast to previously reported results, no IAP transcripts were detected in either F9 cells or two pluripotent cell lines tested. Instead, IAP transcripts as well as IAPs were abundant in the PE-like cells PYS-2 and F9AcCl 9 and in retinoic acid-induced F9 cells but not in the other differentiated cell types of teratocarcinoma origin which were examined. A comparison of the nucleotide sequences of the three IAP cDNA clones with a genomically integrated proviral sequence (MIA14) demonstrated heterogeneity in both length and sequence among the clones. The position of the poly(A) addition site was determined to be 15 nucleotides from the proposed poly(A) addition signal and to occur after the sequence CAGA, not CA, as previously proposed. Length heterogeneity was greatest in a region of TC repeats 80 base pairs 5' to the poly(A) addition site. Additionally, the putative TATAA box found in MIA14 was deleted in the cDNA clones and in the long terminal repeat regions from two other genomic clones examined. The heterogeneity evident among the cDNA clones further demonstrated that at least two distinct IAP genes are activated during differentiation. An analysis of the rate of transcription in isolated nuclei indicated that the activation of expression of IAP genes in PE-like cells is the result of transcriptional regulation. Together, these observations suggest that the modulation of IAP transcription is regulated autonomously rather than by the fortuitous integration of an IAP sequence adjacent to a developmentally regulated cellular gene.
Sequences from sea urchin TU transposons are conserved among multiple eucaryotic species, including humans.
Authors: Liebermann D , Hoffman-Liebermann B , Troutt A B , Kedes L , Cohen S N
Published: September 2015

Sequences homologous to various structural domains of the Strongylocentrotus purpuratus TU family of transposons are present in sea urchin species closely related to S. purpuratus and were found in close proximity to each other in linkage patterns that differed for different species. Sequence homologs of the inverted repeat outer domain (IVR-OD) segment were, in addition, present in a sea urchin related only distantly to S. purpuratus and in all other eucaryotic organisms surveyed. In humans, a polymorphic hybridization pattern was seen for genomic DNA obtained from different individuals. Sequence comparisons revealed that repeated sequence motifs similar to those making up the 15-base-pair direct repeat unit of the IVR-OD domain of the TU elements exist in the IVRs of transposons identified in Drosophila melanogaster and maize and in the transcription control regions of certain eucaryotic viral and cellular genes. The remarkable evolutionary conservation of IVR-OD homologs may reflect a biological role for these sequences in DNA transposition, the regulation of gene expression, or both.
Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae.
Authors: Mann C , Davis R W
Published: September 2015

The CEN4 sequences from chromosome 4 that impart mitotic stability to autonomously replicating (ARS) plasmids in yeast cells have been localized to a 1,755-base-pair (bp) fragment. This fragment could be cut in half to give two adjacent, nonoverlapping fragments, that each contained some mitotic stabilization sequences. One of the half-fragments worked as efficiently as the larger fragment from which it was derived, while the other half provided a much poorer degree of mitotic stabilization. Sequencing of 2,095 bp of DNA including this region revealed the presence of a centromere consensus sequence, elements I, II, and III (M. Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235-244, 1982), in the half-fragment providing high levels of mitotic stability. The poorly stabilizing half-fragment did not contain any obvious sequence homologies to other centromere sequences. Deletion analysis of the 1,755-bp fragment indicated that removal of the 14-bp element I plus 16 of the 82 bp of element II impaired mitotic stability. Removal of elements I and II eliminated the mitotic stability provided by the consensus sequence.
Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences.
Authors: Andersen R D , Birren B W , Taplitz S J , Herschman H R
Published: September 2015

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.
Liver-specific expression of a Qa-encoded class I gene is associated with DNA hypomethylation.
Authors: Miyada C G , Wallace R B
Published: September 2015

DNA methylation of two murine major histocompatibility complex (H-2) class I genes was examined in hybridizations to MspI and HpaII chromosomal DNA restriction digests. Q10, which exhibits liver-specific expression, and H-2Kb, a transplantation antigen gene, were examined in liver, spleen, thymus, and cell-line DNAs. Unmethylated Q10 gene sequences were detected only in the liver, whereas the H-2Kb gene was unmethylated in all tissues examined.
Isolation of chicken cellular DNA sequences with homology to the region of viral oncogenes that encodes the tyrosine kinase domain.
Authors: Foster D A , Levy J B , Daley G Q , Simon M C , Hanafusa H
Published: September 2015

A library of chicken genomic DNA was screened for sequences that could hybridize to a cloned DNA fragment containing the transforming gene (v-fps) of Fujinami sarcoma virus. In addition to c-fps, two unique chicken cellular DNA sequences were isolated that hybridized weakly to v-fps. These sequences hybridized with many other viral oncogenes encoding tyrosine kinases. Sequence analysis of the region where homology was detected revealed a region that is highly conserved among the tyrosine kinases both at the nucleotide and amino acid levels. Although we were unable to detect expression of either chicken cellular DNA sequence in a variety of avian tissues, the data suggest the existence of additional members of the tyrosine kinase gene family. Screening genomic libraries for sequences that hybridize weakly to functional regions of other genes may prove useful for the isolation and characterization of additional members of other gene families.
Regulation of insulin mRNA abundance and adenylation: dependence on hormones and matrix substrata.
Authors: Muschel R , Khoury G , Reid L M
Published: September 2015

The insulin mRNA levels of rat insulinoma cell lines increased six- to eightfold, and the cells entered a transient state of growth arrest when they were cultured in serum-free, hormonally defined medium and on an extract of extracellular matrix derived from a basement membrane-secreting tumor line, EHS. Insulinoma cultures in growth arrest responded to glucose with a two- to threefold increase in insulin secretion associated with an insulin mRNA that contained a poly(A) tail that was 120 to 140 bases longer than normal.
Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.
Authors: Bergman L W , Stranathan M C , Preis L H
Published: September 2015

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.
Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.
Authors: Kelekar A , Cole M D
Published: September 2015

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.
Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene.
Authors: Sive H L , Heintz N , Roeder R G
Published: September 2015

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.
Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.
Authors: Johnston R N , Feder J , Hill A B , Sherwood S W , Schimke R T
Published: September 2015

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.
Formation of the transcription initiation complex on mammalian rDNA.
Authors: Kato H , Nagamine M , Kominami R , Muramatsu M
Published: September 2015

Steps for the formation of transcription initiation complex on the human rRNA gene (rDNA) in vitro were analyzed with partially purified transcription factors and RNA polymerase I. The reaction requires at least two factors besides RNA polymerase I for maximal efficiency. Preincubation and short-pulse analyses of the accurate transcripts revealed the following steps. First, the species-dependent factor, designated TFID, bound to the rDNA template, forming a preinitiation complex (PIC-1) which was resistant to a moderate concentration (0.015 to 0.02%) of Sarkosyl. Other factors, designated TFIA and RNA polymerase I, were then added to convert it to the final preinitiation complex PIC-3. This complex incorporated the first two nucleoside triphosphates of the starting site to complete the initiation complex (IC), which was resistant to a high concentration (0.2%) of Sarkosyl. Binding of TFID was rate limiting in the overall initiation reaction in vitro. Together with the kinetics of incorporation, the results are interpreted to mean that TFID, one bound, remains complexed with rDNA together with TFIA as the PIC-2 for many rounds of transcription by RNA polymerase I. Thus, the formation of PIC-2 may be a prerequisite for the stable opening of rDNA for transcription in vivo.
Bidirectional promoter elements of simian virus 40 are required for efficient replication of the viral DNA.
Authors: Hertz G Z , Mertz J E
Published: September 2015

Mutants of simian virus 40 (SV40) lacking parts of the 72- and 21-base-pair repeat regions were made deficient in large T antigen by recombination with dlA 4000, a mutant containing a frameshift deletion near the amino terminus of the T antigen genes. These double mutants were transfected into COS cells, and the amounts of replicated viral DNA were measured at various times thereafter. It was found that deletion of either the 72- or 21-base-pair repeat region did not significantly reduce the accumulation of viral DNA. However, cells transfected with mutants lacking both of these promoter elements accumulated 100-fold less viral DNA than cells transfected with wild-type SV40. This indicates that the 72- and 21-base-pair repeat regions are each sufficient for supplying a function required for efficient replication of SV40 DNA. In addition, the ability of either of these regions to support efficient replication was gradually reduced as the number of promoter elements within each was decreased. Since the 72- and 21-base-pair repeat regions bidirectionally induce transcription, our results indicate that bidirectional promoter elements play a role in the replication of viral DNA. However, fewer of these elements are required for efficient replication than for efficient transcription.
Overproduction of protein p53 contributes to simian virus 40-mediated transformation.
Authors: Michalovitz D , Eliyahu D , Oren M
Published: September 2015

The possible involvement of p53 overproduction in simian virus 40 (SV40)mediated transformation was studied by using the rat embryo fibroblast focus formation assay. Transformation by wild-type SV40 was enhanced two- to threefold by cotransfection of a plasmid overexpressing mouse p53. More significantly, such a plasmid could partially complement a transformation-defective deletion mutant of SV40. Hence, the ability of SV40 T antigen to induce high p53 levels may indeed be directly relevant to the viral transforming potential.
Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.
Authors: Neigeborn L , Schwartzberg P , Reid R , Carlson M
Published: September 2015

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.
Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system.
Authors: Ahn B Y , Livingston D M
Published: September 2015

Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts. To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3- alleles were placed in a vector. Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3- allele. Many of these events included coconversions in which more than one marker along the allelic sequence was replaced. The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted. From these results the average length of conversion was determined to be approximately 0.5 kilobase. Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers. Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged. The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.
Chromosome-mediated gene transfer of multidrug resistance.
Authors: Gros P , Fallows D A , Croop J M , Housman D E
Published: September 2015

Multidrug resistance can be transferred from drug-resistant LZ Chinese hamster cells to drug-susceptible mouse LTA cells by chromosome-mediated gene transfer. Analysis of genomic DNA demonstrated the transfer of multiple copies of a DNA domain which is amplified in the donor multidrug-resistant cells. The transfer of 10 to 15 copies of the Chinese hamster gene was sufficient to produce a multidrug-resistant phenotype. Chromosome transferents exhibited overexpression of an mRNA of approximately 5 kilobases which has previously been demonstrated to be encoded by the amplified DNA domain of the donor LZ cells. Phenotypic analysis of individual clones selected in adriamycin showed the resistance to be pleiotropic. All clones tested demonstrated similar levels of cross-resistance to the drugs daunorubicin and colchicine. These results indicate that the DNA sequences transferred confer the complete multidrug-resistant phenotype on recipient cells and suggest that multidrug resistance is due to overexpression of the protein encoded by the 5-kilobase mRNA.
Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression.
Authors: Friedman J M , Babiss L E , Clayton D F , Darnell J E
Published: September 2015

Recombinant adenoviruses were constructed in which the viral E1A gene was deleted and the E1B promoter was replaced by the rat albumin, mouse beta-major globin, or mouse immunoglobulin heavy-chain promoter. After infection of human or rat hepatoma cells, E1B-containing mRNAs could be detected only from the virus containing the albumin promoter. Conversely, only the immunoglobulin promoter was active in virus-infected myeloma cells. However, in hepatoma cells transcription from the albumin promoter in the virus was much less than that of the endogenous cellular albumin gene or of other viral genes. In primary mouse hepatocytes endogenous albumin gene transcription was high immediately after plating but declined within 24 h. Expression of the albumin promoter in the virus paralleled that of the cellular albumin gene. From these results it appears that cell-specific expression of albumin depends on the presence of tissue-specific trans-acting factors, but the presence of such factors does not suffice for a maximal rate of transcription, a conclusion that requires direct comparison within a differentiated cell of a newly introduced and preexisting active cell gene.
Microdissection of and microcloning from the short arm of human chromosome 2.
Authors: Bates G P , Wainwright B J , Williamson R , Brown S D
Published: September 2015

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.
Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms.
Authors: Struhl K
Published: September 2015

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.
Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.
Authors: James T C , Elgin S C
Published: September 2015

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.