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1.
Clin Liver Dis ; 26(3): 403-420, 2022 08.
Article in English | MEDLINE | ID: mdl-35868682

ABSTRACT

Hepatitis B and hepatitis C are a global burden and underscore the impact of preventable acute and chronic diseases on personal as well as population level health. Caring for pediatric patients with hepatitis B and C requires a deep understanding of the pathophysiology of viral processes. Insight into the epidemiology, transmission, and surveillance of these infections is critical to prevention and therapy. Extensive research in recent years has created a growing number of treatments, changing the landscape of the medical field's approach to the viral hepatitis pandemic.


Subject(s)
Hepatitis B , Hepatitis C , Hepatitis, Viral, Human , Child , Hepacivirus , Hepatitis B/epidemiology , Hepatitis B/prevention & control , Hepatitis C/epidemiology , Humans
2.
Sci Total Environ ; 646: 245-256, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30055487

ABSTRACT

A reconnaissance project completed in 2009 identified intersex and elevated plasma vitellogenin in male smallmouth bass inhabiting the Missisquoi River, VT. In an attempt to identify the presence and seasonality of putative endocrine disrupting chemicals or other factors associated with these observations, a comprehensive reevaluation was conducted between September 2012 and June 2014. Here, we collected smallmouth bass from three physically partitioned reaches along the river to measure biomarkers of estrogenic endocrine disruption in smallmouth bass. In addition, polar organic chemical integrative samples (POCIS) were deployed to identify specific chemicals associated with biological observations. We did not observe biological differences across reaches indicating the absence of clear point source contributions to the observation of intersex. Interestingly, intersex prevalence and severity decreased in a stepwise manner over the timespan of the project. Intersex decreased from 92.8% to 28.1%. The only significant predictor of intersex prevalence was year of capture, based on logistic regression analysis. The mixed model of fish length and year-of-capture best predicted intersex severity. Intersex severity was also significantly different across late summer and early spring collections indicating seasonal changes in this metric. Plasma vitellogenin and liver vitellogenin Aa transcript abundance in males did not indicate exposure to estrogenic endocrine disrupting chemicals at any of the four sample collections. Analysis of chemicals captured by the POCIS as well as results of screening discrete water samples or POCIS extracts did not indicate the contribution of appreciable estrogenic chemicals. It is possible that unreported changes in land-use activity have ameliorated the problem, and our observations indicate recovery. Regardless, this work clearly emphasizes that single, snap shot sampling for intersex may not yield representative data given that the manifestation of this condition within a population can change dramatically over time.


Subject(s)
Bass/physiology , Endocrine Disruptors/toxicity , Environmental Monitoring , Water Pollutants, Chemical/toxicity , Animals , Biomarkers/metabolism , Disorders of Sex Development/chemically induced , Male , Rivers , Seasons
3.
Ecotoxicol Environ Saf ; 124: 50-59, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26454754

ABSTRACT

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.


Subject(s)
Bass , Disorders of Sex Development , Fish Diseases , Animals , Bass/blood , Bass/metabolism , Cell Line , Disorders of Sex Development/blood , Disorders of Sex Development/metabolism , Disorders of Sex Development/pathology , Disorders of Sex Development/veterinary , Endocrine Disruptors , Estrogens/metabolism , Fish Diseases/blood , Fish Diseases/metabolism , Fish Diseases/pathology , Lakes , Male , New England , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Rivers , Seasons , Testis/pathology , Vitellogenins/blood , Yeasts/genetics , Yeasts/metabolism
4.
Environ Monit Assess ; 186(10): 6471-91, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24934131

ABSTRACT

Fishes were collected at 16 sites within the three major river drainages (Delaware, Susquehanna, and Ohio) of Pennsylvania. Three species were evaluated for biomarkers of estrogenic/antiandrogenic exposure, including plasma vitellogenin and testicular oocytes in male fishes. Smallmouth bass Micropterus dolomieu, white sucker Catostomus commersonii, and redhorse sucker Moxostoma species were collected in the summer, a period of low flow and low reproductive activity. Smallmouth bass were the only species in which testicular oocytes were observed; however, measurable concentrations of plasma vitellogenin were found in male bass and white sucker. The percentage of male bass with testicular oocytes ranged from 10 to 100%, with the highest prevalence and severity in bass collected in the Susquehanna drainage. The percentage of males with plasma vitellogenin ranged from 0 to 100% in both bass and sucker. Biological findings were compared with chemical analyses of discrete water samples collected at the time of fish collections. Estrone concentrations correlated with testicular oocytes prevalence and severity and with the percentage of male bass with vitellogenin. No correlations were noted with the percentage of male sucker with vitellogenin and water chemical concentrations. The prevalence and severity of testicular oocytes in bass also correlated with the percent of agricultural land use in the watershed above a site. Two sites within the Susquehanna drainage and one in the Delaware were immediately downstream of wastewater treatment plants to compare results with upstream fish. The percentage of male bass with testicular oocytes was not consistently higher downstream; however, severity did tend to increase downstream.


Subject(s)
Environmental Monitoring , Fishes/physiology , Water Pollutants, Chemical/analysis , Agriculture , Animals , Biomarkers , Male , Pennsylvania , Reproduction , Rivers/chemistry , Seasons , Vitellogenins/blood , Water Pollutants, Chemical/toxicity
5.
Insect Biochem Mol Biol ; 33(6): 601-7, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12770578

ABSTRACT

The oxidation of farnesol to farnesal is an important step in insect juvenile hormone (JH) biosynthesis and is mediated by one or more alcohol oxidases located within the minute endocrine gland, the corpus allatum. Because lepidopteran insects have the capacity to produce homologous JH structures, the substrate selectivity of farnesol oxidase was examined by determining the ability of several terpenol homologs to inhibit farnesol oxidation in moths. Results utilizing corpora allata homogenates from larval, adult, and embryonic Manduca sexta indicate that increased steric bulk at the C-3 position of the sesquiterpenol chain is detrimental to inhibitory potency. Triethylhomofarnesol (1h), which is precursor to JH 0 and therefore a physiologically important metabolite of M. sexta embryos, was found to be a poor inhibitor of farnesol oxidation but was oxidized in almost same amount as farnesol. This data indicate that farnesol oxidase of the corpus allatum plays a limited role in controlling JH homolog production in moths, and suggests that another oxidative enzyme, which is present at early stages of moth development, is involved in JH homolog construction.


Subject(s)
Alcohol Oxidoreductases/metabolism , Farnesol/metabolism , Juvenile Hormones/biosynthesis , Manduca/metabolism , Animals , Carbon Radioisotopes , Corpora Allata/metabolism , Farnesol/analogs & derivatives , Female , Inhibitory Concentration 50 , Juvenile Hormones/chemistry , Larva/metabolism , Manduca/enzymology , Oxidation-Reduction , Substrate Specificity , Terpenes/chemistry , Terpenes/pharmacology
6.
Insect Biochem Mol Biol ; 31(2): 171-8, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11164339

ABSTRACT

The oxidation of farnesol to farnesoic acid is a key step in insect juvenile hormone biosynthesis. We herein present preliminary characterization of the enzyme-catalyzed oxidation of farnesol to farnesal in larval corpora allata homogenates of the tobacco hornworm, Manduca sexta. This conversion, which is highly substrate specific, has a K(m) apparent of 1 microM and a pH optimum between 6 and 7. Results from chemical modification experiments indicate that the enzyme possesses an active site tyrosine residue. Although farnesol oxidation in adult M. sexta corpora allata homogenates was previously identified as being catalyzed by a dehydrogenase, the corresponding conversion in larvae is not effected by the addition of nicotinamide cofactors. Instead, enzymatic activity is slightly enhanced by the addition of FAD, decreases when incubations are performed anaerobically, and is completely inhibited when either sodium dithionite or glucose oxidase is added. Although the effect of various additives suggests that the oxidation of farnesol to farnesal does not require a metal redox center, 1,10-phenanthroline (but not 4,7-phenanthroline) is a weak irreversible inhibitor of farnesol oxidation (IC(50)=11 mM). The addition of exogenous metals (Fe2+, Cu2+, Ni2+, and Co2+) caused differential effects on farnesol metabolism, with Cu2+ being highly inhibitory. Taken together, this data suggests that the oxidation of farnesol to farnesal in larval corpora allata is mediated by a specific oxygen-dependent enzyme, perhaps a flavin and/or iron-dependent oxidase.


Subject(s)
Alcohol Oxidoreductases/metabolism , Farnesol/metabolism , Juvenile Hormones/biosynthesis , Animals , Binding Sites , Corpora Allata/metabolism , Manduca/metabolism , NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases , Niacinamide/metabolism , Oxidation-Reduction , Tyrosine/metabolism
7.
Biol Reprod ; 62(5): 1360-9, 2000 May.
Article in English | MEDLINE | ID: mdl-10775188

ABSTRACT

We have isolated the full-length coding sequence for mouse KIFC5A (kinesin family c-terminal 5A) cDNA, encoding a motor protein found in the testes. The complete sequence of the KIFC5A cDNA is homologous to a group of carboxyl-terminal motors, including hamster CHO2, human HSET, and mouse KIFC1 and KIFC4. The KIFC5A and KIFC1 cDNAs are nearly identical except for the presence of two additional sequence blocks in the 5'-end of KIFC5A and a number of single base-pair differences in their motor domains. Polymerase chain reaction amplification and sequencing of the 5'-end of KIFC5A identified 3 distinct RNA species in testes and other tissues. Sequence comparison and genetic mapping confirmed the existence of a small multi-gene family in the mouse and suggest possible mechanisms of alternative splicing, genetic duplication, and separate genetic loci in the generation of these motors. In order to examine the possible role of these motors in germ cells of the testes, an antibody to a shared epitope was used to localize this group of proteins to different spermatogenic cell types. These experiments suggest that KIFC5-like motor proteins are associated with multiple microtubule complexes in male germ cells, including the meiotic spindle, the manchette, and the flagella.


Subject(s)
Microtubule-Associated Proteins/genetics , Spermatogenesis/physiology , Amino Acid Motifs , Animals , Antigens/chemistry , Antigens/genetics , Base Sequence , Cloning, Molecular , Cricetinae , Humans , Male , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Homology, Nucleic Acid , Spermatids/chemistry , Spermatids/metabolism , Spindle Apparatus/physiology , Testis/physiology
8.
Diagn Mol Pathol ; 7(2): 76-84, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9785005

ABSTRACT

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400-600 copies of HPV 16, HeLa 229 cells with 10-50 copies of HPV 18, and SiHa cells with 1-2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.


Subject(s)
Biotin/analogs & derivatives , DNA Probes, HPV , DNA, Viral/isolation & purification , In Situ Hybridization/methods , Papillomaviridae/classification , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Tyramine/analogs & derivatives , 3,3'-Diaminobenzidine , Biotin/analysis , Biotinylation , Fluorescein , Formaldehyde , HeLa Cells/virology , Horseradish Peroxidase , Humans , Papillomaviridae/genetics , Papillomaviridae/isolation & purification , Paraffin Embedding , Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and Specificity , Streptavidin , Tissue Fixation , Tumor Cells, Cultured/virology , Tyramine/analysis
9.
Diagn Mol Pathol ; 5(4): 291-6, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8955622

ABSTRACT

The use of oligonucleotide probes with nonisotopic detection systems has made in situ hybridization (ISH) more accessible for use in diagnostic pathology; however, ISH using formalin-fixed, paraffin-embedded tissues remains much more sensitive when performed with radioactively labeled probes compared with nonisotopic reporter systems, especially using oligonucleotide probes. We investigated the effects of microwave pretreatment on the detection of RNA and DNA in formalin-fixed, paraffin-embedded tissues in an attempt to improve the sensitivity of ISH with digoxigenin-labeled probes. Titration of normal tissues with oligonucleotide probe cocktails for albumin, prolactin, chromogranin (Cg) A and B mRNAs and a cDNA probe for JC virus showed a significant increase in sensitivity with the use of microwave treatment step rather than heating at 70 degrees C in 2 x SSC for 30 min. Analysis of various solutions used for microwaving showed that 10 mM citrate buffer was more effective than 2 x SSC, phosphate-buffered saline, tissue unmasking fluid, or water. A 15- to 20-min period of microwaving in an 800-W oven produced optimum results. Analysis of a group of tumors for albumin and CgA and B mRNAs and infected brain biopsies for JC virus DNA showed increased sensitivity of the microwave technique using digoxigenin-labeled probes. These results show that microwave treatment can enhance the detection of mRNA and DNA in formalin-fixed, paraffin-embedded tissues.


Subject(s)
DNA/analysis , In Situ Hybridization , Microwaves , RNA/analysis , Digoxigenin , Humans , In Situ Hybridization/methods , Oligonucleotide Probes , Paraffin Embedding , RNA, Messenger/analysis , Sensitivity and Specificity , Tissue Fixation
10.
Mol Biol Cell ; 7(2): 289-305, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8688559

ABSTRACT

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.


Subject(s)
Calcium-Binding Proteins/metabolism , Kinesins/metabolism , Muscle Proteins/metabolism , Testis/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/isolation & purification , DNA , DNA, Complementary , Gene Expression , Kinesins/genetics , Kinesins/isolation & purification , Male , Mammals , Meiosis/physiology , Molecular Sequence Data , Morphogenesis/physiology , Muscle Proteins/genetics , Muscle Proteins/isolation & purification , Rats , Seminiferous Epithelium/metabolism , Sequence Homology, Amino Acid
11.
Curr Opin Neurobiol ; 5(5): 551-8, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8580705

ABSTRACT

The fact that multiple microtubule-based motors exist in brain inevitably raises questions about their function. Transcripts for at least seven kinesin superfamily genes and even more dynein heavy chain genes have been detected in brain cDNA libraries. The challenge now is to match their gene products to specific functions in cells of the nervous system. Recent studies have attempted to establish a function for each microtubule motor by using recombinant protein and immunochemical approaches.


Subject(s)
GTP Phosphohydrolases/metabolism , Kinesins/metabolism , Microtubules/metabolism , Nervous System Physiological Phenomena , Animals , Dynamins
12.
Proc Natl Acad Sci U S A ; 90(12): 5623-7, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-8516310

ABSTRACT

Chromatin becomes reorganized during mitosis each cell cycle. To identify genes potentially involved in these supramolecular events, we have used a colony-color assay to screen temperature-sensitive mutants of Saccharomyces cerevisiae. When a sequence that mediates attachment to the nuclear matrix in vitro was inserted into the GAL1 promoter of a lacZ fusion gene, beta-galactosidase synthesis was inhibited. This observation permitted screening for temperature-sensitive-inducible mutants on 5-bromo-4-chloro-3-indolyl beta-D-galactoside plates. Only 1 of 20 complementation groups of newly isolated mutants exhibited temperature-sensitive inducibility for the matrix association region but not for control CEN3 or STE6 inserts--a cmd1 mutant in which the last 7 amino acids of calmodulin were truncated by an ochre termination codon. Another mutant (smi1) exhibited a rare phenotype at the nonpermissive condition, which included S phase and budding arrest. We cloned and sequenced the SMI1 gene, which encodes a 57-kDa polypeptide with evolutionarily conserved epitope(s) found in mammalian cell nuclei. Thus, we provide evidence for involvement of calmodulin and another conserved protein in the in vivo binding of a matrix association region.


Subject(s)
Calmodulin/metabolism , Cell Nucleus/metabolism , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Antibodies , Base Sequence , Biological Evolution , Cloning, Molecular , Conserved Sequence , Genes, Fungal , Genetic Complementation Test , Mammals , Molecular Sequence Data , Mutagenesis , Nuclear Matrix/metabolism , Nuclear Proteins/metabolism , Peptides/chemical synthesis , Peptides/immunology , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Sequence Homology, Amino Acid , Temperature , Transcription Factors , beta-Galactosidase/biosynthesis
14.
Proc Natl Acad Sci U S A ; 86(14): 5497-501, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2546156

ABSTRACT

A family of A + T-rich sequences termed MARs ("matrix association regions") mediate chromosomal loop attachment. Here we demonstrate that several MARs both specifically bind and contain multiple sites of cleavage by topoisomerase II, a major protein of the mitotic chromosomal scaffold. Interestingly, "hotspots" of enzyme cutting occur within the MAR of the mouse immunoglobulin kappa-chain gene at the breakpoint of a previously described chromosomal translocation. Since topoisomerase II can mediate illegitimate recombination in prokaryotes, we explored further the possibility that MARs might be targets for this process in eukaryotes. We found that a MAR had been deleted from one of the two rabbit immunoglobulin kappa-chain genes and that MARs reside next to a long interspersed repetitive element within the recombination junction of a human ring chromosome 21. These results, taken together with other accounts of nonhomologous recombination, lead to the proposal that a dysfunction of MARs is illegitimate recombination.


Subject(s)
Chromosome Mapping , DNA Topoisomerases, Type II/metabolism , Genes, Immunoglobulin , Immunoglobulin kappa-Chains/genetics , Recombination, Genetic , Animals , Base Sequence , Cloning, Molecular , DNA, Recombinant/metabolism , Drosophila/enzymology , HeLa Cells/enzymology , Humans , Mice , Molecular Sequence Data , Rabbits , Restriction Mapping
15.
Genome ; 31(2): 503-9, 1989.
Article in English | MEDLINE | ID: mdl-2561108

ABSTRACT

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.


Subject(s)
Chromosomes/ultrastructure , DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Animals , Base Sequence , Binding Sites , Genes, Immunoglobulin , Immunoglobulin kappa-Chains/genetics , Mice , Molecular Sequence Data , Nuclear Matrix/metabolism , Protein Binding , Recombination, Genetic
16.
Mol Cell Biol ; 6(12): 4734-41, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3025668

ABSTRACT

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.


Subject(s)
Poly A/metabolism , Simian virus 40/genetics , Transcription, Genetic , Adenosine Triphosphate/metabolism , Adenoviruses, Human/genetics , Base Sequence , Cell Nucleus/metabolism , RNA, Viral/metabolism , Simian virus 40/metabolism , Templates, Genetic
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