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J W Fewell

Publications and source records attributed to J W Fewell.

15 recordsLinked to original sources
PubMed1996

Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium.

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.

3T3 Cells↗
PubMed1993

EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.

Adenoviruses, Human↗
PubMed1993

Selective expression of intracisternal A-particle genes in established mouse plasmacytomas.

Mouse plasmacytomas generally express higher levels of RNA transcripts from endogenous intracisternal A-particle (IAP) proviral elements than do lipopolysaccharide-stimulated normal lymphocytes. Lymphocytes express a limited and highly characteristic set of IAP elements (lymphocyte-specific [LS] elements). In this study, we examined whether LS elements are expressed at higher levels after transformation of the cells and/or whether new IAP elements are activated. The IAP elements expressed in plasmacytoma MPC11 were characterized by sequence analysis of 22 cDNA clones. The long terminal repeats (LTRs) of the tumor cDNAs proved to be highly related in sequence. None of the clones was of the LS cDNA type. The MPC11 LTRs were five- to sixfold more active than an LS cDNA LTR when tested for promoter activity by transfection into plasmacytoma cells. The LTRs of the tumor-derived cDNAs contained a canonical ATF core sequence (ATF-PC), while the LS cDNAs contained an altered sequence (ATF-LS). An ATF-PC oligonucleotide probe detected multiple IAP transcripts on Northern (RNA) blots of RNA from several plasmacytoma but gave no reaction with RNA from stimulated B lymphocytes. In contrast, an ATF-LS probe detected higher levels of RNA in lymphocyte than in tumor RNAs. Thus, expression of IAP elements in transformed B cells is selective for a different set of regulatory sequence variants than those expressed in normal B cells. Other oligonucleotide probes representing LS- and PC-specific sequence variants detected multiple common hypomethylated IAP proviral loci in three independently derived plasmacytomas. Overall, the results show that established plasmacytomas exhibit a characteristic pattern of IAP proviral hypomethylation and regulatory sequence selection.

Animals↗
PubMed1992

Selective activation of a discrete family of endogenous proviral elements in normal BALB/c lymphocytes.

Intracisternal A-particle (IAP) proviral elements are abundant and widely dispersed in the mouse genome. IAP-related transcripts have been detected in normal mouse tissues where expression is under genetic control. In this study, we sought to determine whether IAP expression in BALB/c thymus and lipopolysaccharide-stimulated B cells was due to selective or indiscriminate activation of IAP elements. cDNA libraries were prepared from each source. A total of 86 IAP cDNA clones were isolated from both libraries, and 37 of these were sequenced over a common 0.7- to 1.0-kb region of the IAP genome that included the 3' long terminal repeat (LTR). Three highly related families of elements were found to be expressed in the two cell types examined. All of the related elements had a distinctive U3 regulatory region. Thirteen individual IAP proviral elements were distinguished on the basis of sequence differences within the R region of the LTR. Hybridization of genomic DNA with element-specific oligonucleotide probes confirmed the presence of a restricted number of proviral copies in the lymphocyte-specific family of elements. Most of these copies were found to be methylated in the lymphocyte DNA, but at least seven were hypomethylated in their 5' LTRs. This study shows that activation of IAP elements in normal normal mouse lymphocytes is highly selective. Activation is probably a function of both sequence specificity and methylation status of the proviral LTR.

Animals↗
PubMed1989

Characterization of amplified intracisternal A-particle elements encoding integrase.

Type IIB intracisternal A-particle (IAP) elements have undergone marked amplification and transposition in the genomic DNA of some mouse myelomas. We have made a cDNA library from one such myeloma, MOPC 315, to determine whether some property of the elements themselves has a role in this process. Sequencing of several type IIB cDNAs and one genomic type IIB IAP element has shown that they are nearly identical (greater than 99%) and contain 2 open reading frames (ORFs). ORF2 is capable of encoding the IAP integrase, an enzyme which catalyzes integration of proviral DNA into the genome. An antiserum to a synthetic peptide based on the IAP integrase gene sequence reacted with ORF2 product expressed in bacteria as a fusion protein, and detected a 47 kDa protein, predicted from the size of ORF2, in myeloma cell fractions by Western blotting.

Amino Acid Sequence↗
PubMed1986

Glucose induces intracisternal type A retroviral gene transcription and translation in pancreatic beta cells.

C57BL/KsJ (BKs) and CBA/J, but not C57BL/6J (B6) mice are susceptible to the diabetogenic action of the obesity gene, "diabetes" (db). BKs and CBA/J, but not B6 mice, constitutively express intracisternal type A particles (IAP), an endogenous class of retrovirus, in beta cells and in cortical thymocytes. IAP genetic expression in these cell types included production of the group-specific antigen, p73, as well as higher-molecular mass p73-related antigens (p114-120). We used islet culture techniques to show that both transcription and translation of IAP genomes in beta cells in enhanced by glucose. Maintenance of CBA/J islets for 48 h in 16.5 mM glucose-containing medium effected a fivefold induction of IAP protein synthesis in comparison to islets cultured in low- (5.5 mM) glucose medium. Analysis of RNA from 16.5 mM glucose-cultured islets revealed induction of 7.2 and 5.4 kbp transcripts known to code for p73 and the p114-120 polypeptides, respectively. This induction in CBA/J islets was 10-15-fold on a tissue basis, and 5-7-fold on an RNA basis. Glucose induction of preproinsulin mRNA levels was also analyzed in the same samples. Islets cultured in 16.5 mM glucose showed an eightfold higher level on a tissue basis, and a fourfold increase in terms of total recovered RNA. Comparison of these glucose-inducible parameters in islets isolated from the diabetes-susceptible BKs strain vs. the resistant B6 strain revealed that expression of the group-specific retroviral p73 antigen was limited to BKs beta cells. This inbred strain control of p73 expression was also found in cortical thymocytes, with B6 thymocytes producing a 117 kD component to the exclusion of p73, while both components were expressed in thymocytes from normal BKs mice. In comparison to normal BKs males, thymocytes from four week-old genetically diabetic (db/db) BKs males showed no change in labeling of p117, but showed a sharply diminished incorporation into p73. This suggested that accelerated thymic involution characteristic of db/db mice may entail selective elimination of p73-producing cells. The possibility that glucose-stressed BKs pancreatic beta cells are marked for autoimmune elimination by the elaboration of p73 or other IAP-related proteins is discussed.

Animals↗
PubMed1985

Intracisternal A-particle gene expression in normal mouse thymus tissue: gene products and strain-related variability.

Intracisternal A-particle (IAP)-specific sequences were 5- to 10-fold enriched in polyadenylated RNA from BALB/cJ thymus as compared with RNAs from liver, spleen, and kidney. The major transcripts of 7.2 and 5.4 kilobases were the same size as those found in an IAP-rich neuroblastoma cell line. The absolute levels and proportions of these transcripts varied in thymuses from mice of different inbred strains. With antiserum prepared against p73, the main IAP structural protein, several size classes of IAP-related proteins were immunoprecipitated from extracts of thymus cells incubated with [35S]methionine; these included p73 itself and a group of polypeptides in the size range of 114 to 120 kilodaltons (p114-p120). The inbred strains showed marked characteristic differences in the electrophoretic patterns of their IAP-related proteins. Earlier studies showed that the 7.2-kilobase RNA from neuroblastoma IAPs coded for p73 in a cell-free translation system. Correlations between the RNA and protein patterns in thymuses of the different inbred strains indicated that 5.4-kilobase RNA gives rise to the p114-p120 polypeptides. Metabolically labeled p120 was found to include methionine-containing tryptic peptides of p73 plus additional peptides consistent with its larger size. In vivo labeling kinetics showed that the p114-p120 polypeptides were not major precursors of p73 in intact neuroblastoma cells. This study shows that IAP gene expression in mouse thymus is genetically determined and that a novel class of IAP-related polypeptides can be expressed independently of the major particle structural protein.

Animals↗
PubMed1984

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

Acetyltransferases↗
PubMed1983

DNA polymerase and simian virus 40 infection of resting monkey cells: induction of aphidicolin resistant alpha-polymerase.

Tightly confluent monkey cell lines BSC-1 and CV-1 held in stale medium for several days exhibited an extremely low level of thymidine incorporation into cellular DNA. Yet, these cells contained a level of alpha-polymerase equal to about 15% of the level in rapidly dividing cells, and they still were capable of supporting replication of SV40 DNA. SV40 infection and culture in stale medium resulted in a four-fold induction of alpha-polymerase in CV-1 cells, whereas no change in alpha-polymerase level was observed in BSC-1 cells. Characterization of alpha-polymerase partially purified from infected CV-1 cells revealed that 80-90% of the enzyme activity was aphidicolin resistant. SV40 DNA replication in resting CV-1 cells, however, was aphidicolin sensitive. SV40 infection of resting CV-1 cells may induce an aphidicolin-resistant enzyme or lead to a modified alpha-polymerase species.

Animals↗