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Expression of an endogenous murine leukemia virus-related proviral sequence is androgen regulated and primarily restricted to the epididymis/vas deferens and oviduct/uterus.
Cornwall GA, Orgebin-Crist MC, Hann SR
(1992) Biol Reprod 47: 665-75
MeSH Terms: Androgens, Animals, Base Sequence, DNA, Viral, Epididymis, Female, Gene Expression, Genes, Viral, Genitalia, Leukemia Virus, Murine, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Ovarian Follicle, Proviruses, Uterus, Vas Deferens
Show Abstract · Added March 5, 2014
A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.
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18 MeSH Terms
Infectious defective interfering particles of VSV from transcripts of a cDNA clone.
Pattnaik AK, Ball LA, LeGrone AW, Wertz GW
(1992) Cell 69: 1011-20
MeSH Terms: Base Sequence, Blotting, Northern, Cells, Cultured, DNA, Defective Viruses, In Vitro Techniques, Molecular Sequence Data, Oligodeoxyribonucleotides, RNA Replicase, RNA, Viral, Transfection, Vesicular stomatitis Indiana virus, Viral Interference, Virus Replication
Show Abstract · Added February 3, 2011
The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported. Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones. The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions. No helper VSV was required. Replication occurred at high levels and was assayed by direct biochemical means. An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.
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14 MeSH Terms
Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination.
Gale KC, Osheroff N
(1992) J Biol Chem 267: 12090-7
MeSH Terms: Adenosine Triphosphate, Animals, Autoradiography, Bacteriophage phi X 174, Base Sequence, Blotting, Southern, Cations, Divalent, DNA Topoisomerases, Type II, DNA, Viral, Drosophila melanogaster, Molecular Sequence Data, Nucleic Acid Conformation, Recombination, Genetic, Substrate Specificity
Show Abstract · Added March 5, 2014
Drosophila melanogaster topoisomerase II is capable of joining phi X174 (+) strand DNA that it has cleaved to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction (Gale, K. C. and Osheroff, N. (1990) Biochemistry 29, 9538-9545). In order to investigate potential mechanisms for topoisomerase II-mediated DNA recombination, this intrinsic enzyme activity was further characterized. Intermolecular DNA ligation proceeded in a time-dependent fashion and was concentration-dependent with respect to oligonucleotide. The covalent linkage between phi X174 (+) strand DNA and acceptor molecules was confirmed by Southern analysis and alkaline gel electrophoresis. Topoisomerase II-mediated intermolecular DNA ligation required the oligonucleotide to contain a 3'-OH terminus. Moreover, the reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating phi X174 (+) strand DNA to double-stranded oligonucleotides that contained 5'-overhang, 3'-overhand, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides also could be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.
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14 MeSH Terms
Selective disruption of genes expressed in totipotent embryonal stem cells.
von Melchner H, DeGregori JV, Rayburn H, Reddy S, Friedel C, Ruley HE
(1992) Genes Dev 6: 919-27
MeSH Terms: Alcohol Oxidoreductases, Animals, Base Sequence, Blotting, Northern, Cell Line, Chimera, Female, Genetic Vectors, Kanamycin Kinase, Male, Mice, Molecular Sequence Data, Mutagenesis, Insertional, Phosphotransferases, Promoter Regions, Genetic, Recombinant Fusion Proteins, Stem Cells
Show Abstract · Added May 29, 2014
Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
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17 MeSH Terms
Two promoters in the bovine adrenodoxin gene and the role of associated, unique cAMP-responsive sequences.
Chen JY, Waterman MR
(1992) Biochemistry 31: 2400-7
MeSH Terms: Adrenodoxin, Animals, Base Sequence, Cattle, Cyclic AMP, Cyclic AMP Response Element-Binding Protein, Cycloheximide, DNA-Binding Proteins, Kidney, Liver, Molecular Sequence Data, Promoter Regions, Genetic, Transcription Factors
Show Abstract · Added February 12, 2015
The bovine adrenodoxin gene gives rise to two species of mRNA differing only at their 5'-ends. The synthesis of these two types of mRNA in bovine adrenal cortical cells is regulated transcriptionally in part by ACTH via the action of cAMP. Examination of the 5'-end of the adrenodoxin gene revealed that each mRNA contains sequences derived from a different exon encoding the mitochondrial leader sequence. To define the sequences necessary for the synthesis of these two types of mRNA and to determine if the synthesis of each is driven by a separate promoter, 5'-regions of the adrenodoxin gene were inserted upstream from two different reporter genes and tested for promoter/enhancer regulatory activity by transient transfection into mouse adrenocortical Y1 tumor cells. The results clearly demonstrated that the bovine adrenodoxin gene contains two functional promoters; one, ADXP1, located in the 5'-flanking region gives rise to the minor form of mRNA, and a second, stronger promoter, ADXP2, which maps within intron 1 gives rise to the major form of mRNA. Unique cAMP-responsive sequences were found upstream from each promoter which share no sequence homology to the consensus CRE (cAMP-responsive element). Upon transient expression, the cAMP-responsive sequence associated with the ADXP2 promoter, termed CRS2, confers the cAMP responsiveness to stimulate the transcription of the linked beta-globin reporter gene regardless of whether the adrenodoxin ADXP2 promoter or the beta-globin promoter was utilized.(ABSTRACT TRUNCATED AT 250 WORDS)
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13 MeSH Terms
Distinct biochemical mechanisms for cAMP-dependent transcription of CYP17 and CYP21.
Zanger UM, Kagawa N, Lund J, Waterman MR
(1992) FASEB J 6: 719-23
MeSH Terms: Adrenal Glands, Animals, Base Sequence, Binding Sites, Cattle, Cyclic AMP, Humans, Molecular Sequence Data, Nuclear Proteins, Steroid 17-alpha-Hydroxylase, Steroid 21-Hydroxylase, Transcription, Genetic
Show Abstract · Added February 12, 2015
Optimal steroidogenic capacity in the adrenal cortex is regulated by ACTH via cAMP and involves transcription of the genes encoding the adrenocortical steroid hydroxylases. The microsomal steroid hydroxylases, P45017 alpha and P450C21, are encoded by CYP17 and CYP21, respectively. These genes are thought to have arisen from a common progenitor gene and are coordinately regulated by ACTH. The cAMP responsive sequences (CRS) located in the 5'-flanking regions of these genes are distinct from one another and from known consensus sequences imparting cAMP responsiveness in other genes. The CYP21 CRS binds a putative adrenal-specific nuclear protein. In contrast, the CYP17 CRSI binds a ubiquitous protein that is apparently active only in steroidogenic cells. Thus the ACTH-dependent transcription of these two genes, which have a common evolutionary origin and are coordinately expressed in the adrenal cortex, involves distinct biochemical mechanisms.
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12 MeSH Terms
Functions of the sequences at the ends of the inverted repeats of pseudorabies virus.
Rall GF, Kupershmidt S, Sugg N, Veach RA, Ben-Porat T
(1992) J Virol 66: 1506-19
MeSH Terms: Base Sequence, Blotting, Southern, DNA Mutational Analysis, DNA, Viral, Herpesvirus 1, Suid, Molecular Sequence Data, Regulatory Sequences, Nucleic Acid, Repetitive Sequences, Nucleic Acid, Restriction Mapping
Show Abstract · Added May 20, 2014
Two mutants were constructed to explore the functions of the sequences at the end of the S terminus of pseudorabies virus (PrV). In mutant vYa, 17 bp from the internal inverted repeat, as well as adjacent sequences from the L component, were deleted. In mutant v135/9, 143 bp from the internal inverted repeat (including sequences with homology to the pac-1 site of herpes simplex virus), as well as adjacent sequences from the L component, were deleted. Our aim in constructing these mutants was to ascertain whether equalization of the terminal regions of the S component would occur, whether genome termini that lack either the terminal 17 or 143 bp would be generated as a result of equalization of the repeats (thereby identifying the terminal nucleotides that may include cleavage signals), and whether inversion of the S component would occur (thereby ascertaining the importance of the deleted sequences in this process). The results obtained show the following (i) The removal of the terminal 17 or 143 bp of the internal S component, including the sequences with homology to the pac-1 site, does not affect the inversion of the Us. (ii) The equalization of both the vYa and the v135/9 inverted repeats occurs at high frequency, the terminal repeats being converted and becoming similar to the mutated internal inverted repeat. (iii) Mutants in which the 17 terminal base pairs (vYa) have been replaced by unrelated sequences are viable. However, the 143 terminal base pairs appear to be essential to virus survival; concatemeric v135/9 DNA with equalized, mutant-type, inverted repeats accumulates, but mature virions with such equalized repeats are not generated at high frequency. Since concatemeric DNA missing the 143 bp at both ends of the S component is not cleaved, the terminal 143 bp that include the sequences with homology to the pac-1 site are necessary for efficient cleavage. (iv) v135/9 intracellular DNA is composed mainly of arrays in which one S component (with two equalized inverted repeats both having the deletion) is bracketed by two L components in opposite orientations and in which two L components are in head-to-head alignment.(ABSTRACT TRUNCATED AT 250 WORDS)
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9 MeSH Terms
A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage.
Corbett AH, Zechiedrich EL, Osheroff N
(1992) J Biol Chem 267: 683-6
MeSH Terms: Animals, Base Sequence, DNA, DNA Topoisomerases, Type II, Drosophila melanogaster, Electrophoresis, Polyacrylamide Gel, Microscopy, Electron, Molecular Sequence Data, Nucleic Acid Conformation, Oligonucleotides, Substrate Specificity
Show Abstract · Added March 5, 2014
Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.
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11 MeSH Terms
DNA methylation represses FMR-1 transcription in fragile X syndrome.
Sutcliffe JS, Nelson DL, Zhang F, Pieretti M, Caskey CT, Saxe D, Warren ST
(1992) Hum Mol Genet 1: 397-400
MeSH Terms: Base Sequence, Chorionic Villi Sampling, DNA, Exons, Female, Fetus, Fragile X Mental Retardation Protein, Fragile X Syndrome, Humans, Male, Methylation, Nerve Tissue Proteins, Pedigree, Polymerase Chain Reaction, Pregnancy, RNA-Binding Proteins, Repetitive Sequences, Nucleic Acid, Restriction Mapping, Transcription, Genetic
Show Abstract · Added February 20, 2014
Fragile X syndrome is the most frequent form of inherited mental retardation and segregates as an X-linked dominant with reduced penetrance. Recently, we have identified the FMR-1 gene at the fragile X locus. Two molecular differences of the FMR-1 gene have been found in fragile X patients: a size increase of an FMR-1 exon containing a CGG repeat and abnormal methylation of a CpG island 250 bp proximal to this repeat. Penetrant fragile X males who exhibit these changes typically show repression of FMR-1 transcription and the presumptive absence of FMR-1 protein is believed to contribute to the fragile X phenotype. It is unclear, however, if either or both molecular differences in FMR-1 gene is responsible for transcriptional silencing. We report here the prenatal diagnosis of a male fetus with fragile X syndrome by utilizing these molecular differences and show that while the expanded CGG-repeat mutation is observed in both the chorionic villi and fetus, the methylation of the CpG island is limited to the fetal DNA (as assessed by BssHII digestion). We further demonstrate that FMR-1 gene expression is repressed in the fetal tissue, as is characteristic of penetrant males, while the undermethylated chorionic villi expressed FMR-1. Since the genetic background of the tissues studied is identical, including the fragile X chromosome, these data indicate that the abnormal methylation of the FMR-1 CpG-island is responsible for the absence of FMR-1 transcription and suggests that the methylation may be acquired early in embryogenesis.
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19 MeSH Terms
Cloning of a type I keratin from goldfish optic nerve: differential expression of keratins during regeneration.
Druger RK, Levine EM, Glasgow E, Jones PS, Schechter N
(1992) Differentiation 52: 33-43
MeSH Terms: Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA, Goldfish, Keratins, Molecular Sequence Data, Nerve Regeneration, Optic Nerve, Protein Binding, RNA, Messenger, Sequence Homology, Amino Acid
Show Abstract · Added November 2, 2015
We report the cDNA sequence and predicted amino acid sequence of a novel type I keratin, designated as GK50, and show that keratin expression in the goldfish optic nerve is highly complex. The GK50 protein is one of at least three type I keratins expressed in goldfish optic nerve based on both antibody reactivity and blot-binding to the type II keratin ON3. After optic nerve crush in situ hybridization shows a localized increase in GK50 mRNA expression in the crush zone. This is in contrast to ON3 mRNA which shows a localized increase that is limited to the proximal and distal margins of the crush zone, suggesting a diversity of keratin expression in different cell types of the goldfish optic nerve.
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13 MeSH Terms