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J T Miller

Publications and source records attributed to J T Miller.

At least 19 recordsLinked to original sources
PubMed2001

An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.

We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.

Abatacept↗
PubMed2001

A novel interaction of tRNA(Lys,3) with the feline immunodeficiency virus RNA genome governs initiation of minus strand DNA synthesis.

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)(4) sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

Base Sequence↗
PubMed2001

Molecular phylogenetics of Acacia (Fabaceae: Mimosoideae) based on the chloroplast MATK coding sequence and flanking TRNK intron spacer regions.

The tribe Acacieae (Fabaceae: Mimosoideae) contains two genera, the monotypic African Faidherbia and the pantropical Acacia, which comprise about 1200 species with over 950 confined to Australia. As currently recognized, the genus Acacia is subdivided into three subgenera: subg. Acacia, subg. Aculeiferum, and the predominantly Australian subg. Phyllodineae. Morphological studies have suggested the tribe Acacieae and genus Acacia are artificial and have a close affinity to the tribe Ingeae. Based on available data there is no consensus on whether Acacia should be subdivided. Sequence analysis of the chloroplast trnK intron, including the matK coding region and flanking noncoding regions, indicate that neither the tribe Acacieae nor the genus Acacia are monophyletic. Two subgenera are monophyletic; section Filicinae of subgenus Aculeiferum does not group with taxa of the subgenus. Section Filicinae, eight Ingeae genera, and Faidherbia form a weakly supported paraphyletic grade with respect to subg. Phyllodineae. Acacia subg. Aculeiferum (s. s.) is sister to the grade. These data suggest that characters currently used to differentiate taxa at the tribal, generic, and subgeneric levels are polymorphic and homoplasious in cladistic analyses.

Journal Article↗
PubMed2000

Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes.

The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe(2+) fails to replace Mg(2+) in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.

Base Sequence↗
PubMed1999

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Antineoplastic Agents↗
PubMed1998

Rice (Oryza sativa) centromeric regions consist of complex DNA.

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.

Base Sequence↗
PubMed1998

Mutating a region of HIV-1 reverse transcriptase implicated in tRNA(Lys-3) binding and the consequences for (-)-strand DNA synthesis.

Recently, tRNALys-3 was cross-linked via its anticodon loop to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) between residues 230 and 357 (Mishima, Y., and Steitz, J. A. (1995) EMBO J. 14, 2679-2687). Scanning the surface of this region identified three basic amino acids Lys249, Arg307, and Lys311 flanking a small crevice on the p66 thumb subdomain outside the primer-template binding cleft. To assess an interaction of this region with the tRNA anticodon loop, these p66 residues were altered to Glu or Gln. p66 subunits containing K249Q, K311Q, K311E, and a dual R307E/K311E mutation formed a stable dimer with wild type p51. All mutants showed reduced affinity for tRNALys-3 and supported significantly less (-)-strand DNA synthesis from this primer than the parental heterodimer. In contrast, these variants efficiently synthesized HIV-1 (-)-strand strong-stop DNA from oligonucleotide primers and had minimal effect on RNase H activity, retaining endonucleolytic and directed cleavage of an RNA/DNA hybrid. Structural features of binary RT.tRNALys-3 complexes were examined by in situ footprinting, via susceptibility to 1, 10-phenanthroline-copper-mediated cleavage. Unlike wild type RT, mutants p66(K311Q)/p51 and p66(K311E)/p51 failed to protect the tRNA anticodon domain from chemical cleavage, indicating a significant structural alteration in the binary RT.tRNA complex. These results suggest a crevice in the p66 thumb subdomain of HIV-1 RT supports an interaction with the tRNALys-3 anticodon loop critical for efficient (-)-strand DNA synthesis.

Anticodon↗
PubMed1998

Human prostatic carcinoma cell lines display altered regulation of polyamine transport in response to polyamine analogs and inhibitors.

BACKGROUND: The possibility was investigated that complex homeostatic mechanisms which maintain polyamine pools in prostate-derived tumors may differ from those which are typically seen in other tissues and tumors. METHODS: Growth sensitivity and various regulatory responses were investigated in three human prostate carcinoma cell lines (LNCaP, DU145, and PC-3) treated with the inhibitor of S-adenosylmethionine decarboxylase CGP-48664 or the polyamine analog N1,N11-diethylnorspermine (DENSPM), both of which are currently undergoing phase I clinical trial. RESULTS: Prostate tumor cell lines were all similarly growth-inhibited by the inhibitor CGP-48664 (IC50 values, 1-5 microM at 72 hr), but varied considerably in their sensitivity to DENSPM. The rank-order for cell-line growth inhibition by the analog was DU145 > PC-3 > LNCaP, with IC50 values of 1, 30, and 1,000 microM, respectively. Both compounds depleted intracellular polyamine pools to levels which seemed sufficient to account for inhibition of cell growth. While polyamine enzyme regulatory responses to both CGP-48664 and DENSPM were typical of those seen in other cell types, regulation of polyamine transport differed distinctly. Based on Vmax determinations, LNCaP cells failed to upregulate transport in response to CGP-48664, while PC-3 and LNCaP cells failed to downregulate transport in response to DENSPM. CONCLUSIONS: Relative to other cell lines, polyamine transport in prostate carcinoma cell lines was found to be uniquely insensitive to regulation by polyamines or analogs. Although this did not seem to correlate with growth sensitivity to polyamine analogs in vitro, it should be therapeutically exploitable in in vivo systems.

Adenosylmethionine Decarboxylase↗
PubMed1998

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.

Amino Acid Sequence↗
PubMed1997

Multiple biological roles associated with the Rous sarcoma virus 5' untranslated RNA U5-IR stem and loop.

The 5' untranslated region of Rous sarcoma virus (RSV) RNA is a highly ordered structure involved in multiple processes in the viral replication cycle. One of these structures, referred to as the U5-IR stem, is located immediately upstream of the 5' end of the primer binding site. Disruption of its base pairing results in a decrease in initiation of reverse transcription (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991). In the present study, the length of the U5-IR stem structure has been extended by insertions of different sequences which decrease the efficiency of reverse transcription, in vivo and in vitro. Reverse transcription is rescued partially by placing single-stranded bulges into the middle of the extended duplexes. Nucleotide substitutions or insertions into the loop region of the U5-IR stem also decrease the efficiency of reverse transcription, suggesting that these sequences may specifically interact with reverse transcriptase. Surprisingly, all of the extended stem mutations cause significant RNA packaging defects. In contrast, nucleotide insertions or base substitutions in the U5-IR loop do not affect RNA packaging. These data indicate that the reverse transcription initiation complex and RNA packaging apparatus are influenced by the same region of RSV RNA and that each process is differentially sensitive to changes in sequence and/or secondary structure.

Animals↗
PubMed1995

Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.

The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.

3T3 Cells↗
PubMed1995

Femoral artery catheterization complications: a study of 503 consecutive patients.

This report describes a prospective randomized trial of 503 patients who underwent a cardiac catheterization or interventional procedure at a single institution. In an effort to study femoral complications postprocedure, we evaluated three methods of femoral artery hemostasis as well as 38 variables that were felt to potentially relate to local complications. Only a marginally significant relationship between the hemostasis method and complication rate was found. The factors that contributed to femoral artery complications were: restarting heparin postsheath removal, number of procedures done during one hospitalization, noncompliance of the patient with bedrest after the procedure, number of arterial punctures to initiate the procedure, and preprocedure treatment with corticosteroids.

Adult↗
PubMed1994

Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region.

Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G-protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have identified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signalling, and structural, molecules important in growth control and the regulation of cytoskeletal organization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Regulation of polyamine transport by polyamines and polyamine analogs.

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in Vmax to levels approximately 4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N1, N12-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 microM BESPM, Vmax values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400-500 pmol/10(6) cells--about 15-20% of the total polyamine pool (approximately 2.8 nmol/10(6) cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 microM produced an increase in SPD and SPM Vmax values, whereas concentrations 3 microM and higher produced a marked suppression of these values. Cells treated with 3 microM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15-20%) changes in intracellular polyamine pools.

Animals↗
PubMed1993

Staphylococcal esophagitis causing giant ulcers.

A 29-year-old woman with Hodgkin disease developed odynophagia while receiving chemotherapy. Large esophageal ulcers due to staphylococcal infection of the mucosa were visualized by endoscopy and radiography. This unusual bacterial esophagitis represents another potential cause of giant esophageal ulcerations.

Adult↗
PubMed1992

Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA.

Retroviruses, pararetroviruses, and related retrotransposons generate terminally redundant RNAs by transcription of a template flanked by long terminal repeats in which initiation occurs within the 5' long terminal repeat sequences and 3'-end processing occurs within the 3' long terminal repeat sequences. Processing of avian sarcoma virus RNA is relatively inefficient; approximately 15% of the viral RNA transcripts are read-through products; i.e., they are not processed at the viral poly(A) addition site but at sites in the cellular sequence further downstream. In this report, we show that the efficiency of processing at the viral site is further reduced by deletion of two distant upstream sequences: (i) a 606-nucleotide sequence in the gag gene containing a cis-acting negative regulator of splicing and (ii) a 136-nucleotide sequence spanning the env 3' splice site. The deletion of either or both upstream regions increases the levels of read-through products of both unspliced and spliced viral RNA. In contrast, deletion of the src 3' splice site does not affect the efficiency of processing at the viral poly(A) addition site. The effects on 3'-end processing are not correlated either with distance from the promoter to the poly(A) addition site or with the overall level of viral RNA splicing. Substitution of the avian sarcoma virus poly(A) signal with the simian virus 40 early or late poly(A) signal relieves the requirement for the distant upstream sequences. We propose that cellular factors, which may correspond to splicing factors, bound to the upstream viral sequences may interact with factors bound at the avian sarcoma virus poly(A) signal to stabilize the polyadenylation-cleavage complex and allow for more efficient 3'-end processing.

Animals↗