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At least 217 records · Page 12Linked to original sources

PubMed1994

Sequence analysis of 12 structural genes and a novel non-coding region from mitochondrial DNA of Atlantic cod, Gadus morhua.

We have determined the nucleotide sequences of 12 structural genes from the mitochondrial DNA of Atlantic cod, Gadus morhua. These genes encode the proteins NADH dehydrogenase subunit 2, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, and apocytochrome b, as well as the transfer RNAs tRNA(Ile), tRNA(Gln), tRNA(Met), tRNA(Ser) (UCN), tRNA(Asp), tRNA(Glu), tRNA(Thr) and tRNA(Pro). The apocytochrome b sequences were used to construct a phylogenetic tree revealing the evolutionary divergence between modern bony fishes, sturgeon and sharks. We found that bony fishes display the same slow amino acid substitution rates in the mitochondrial encoded proteins as cartilaginous fishes (sharks). A novel non-coding region of 74 base pairs not found in other fishes where sequence data are available is located between the genes encoding tRNA(Thr) and tRNA(Pro). This region contains both direct and inverted repeat motifs that may function in termination of the H-strand transcript.

Amino Acid Sequence↗

PubMed1999

Endothelial cell DNA transfer and expression using petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system.

The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150-450 V), pulse times (10-40 ms), DNA concentrations (0-20 microg/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.

Bacteriophage T7↗

PubMed2000

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism.

The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.

Base Sequence↗

PubMed2006

Viral gene therapy.

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses. The ability for the delivery of therapeutic genes, made them desirable for engineering virus vector systems. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expression in vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many non-dividing and dividing cell types, but has a limited DNA capacity. This review discusses current and emerging virusbased genetic engineering strategies for the delivery of therapeutic molecules or several approaches for cancer treatment.

Antineoplastic Agents↗

PubMed1997

Understanding species-specific differences in substrate recognition by Escherichia coli and human prolyl-tRNA synthetases.

Class II human prolyl-tRNA synthetase (ProRS) aminoacylates in vitro transcribed human tRNA(Pro) with kinetic parameters that are similar to those previously determined for aminoacylation of Escherichia coli tRNA(Pro) by its cognate synthetase. As in the bacterial system, large decreases in aminoacylation by human ProRS occur upon mutating anticodon positions G35 and G36 of human tRNA(Pro). The N73 'discriminator' base and the first and third base pairs of the acceptor stem vary between the E.coli and human isoacceptor groups. In contrast to the E. coli synthetase, the human enzyme does not appear to recognize these elements, since mutations at these positions do not significantly affect cognate synthetase charging. E. coli ProRS does not cross-aminoacylate human tRNA(Pro), and the bacterial tRNA(Pro) is a poor substrate for the human enzyme. Mutations in both the tRNAs and the synthetases have been made in an effort to identify elements in each system responsible for blocking cross-species aminoacylation. Alignment of all known ProRS primary sequences from different species reveals particularly low overall sequence homology, as well as two distinct groups of enzymes. The sequence divergence between E. coli and human ProRSs helps to explain the species-specific differences in the RNA code for aminoacylation of tRNA(Pro).

Amino Acid Sequence↗

PubMed2001

Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound.

The use of systemic IGF-1 has been shown to attenuate the postburn hypermetabolic response and improve burn wound healing. Local IGF-1 gene therapy, however, promotes re-epithelialization in the burn wound without the side-effects associated with systemic delivery. We tested the hypothesis that these beneficial effects are due to changes in local cytokine production. Adult male Sprague-Dawley rats received a 40% total body surface area full-thickness scald burn and randomly received a subcutaneous injection at the burn wound margin of saline or cationic liposomes containing a IGF-1 cDNA construct. Animals were killed at 1, 4, 7 and 10 days after burn trauma. Skin biopsies at the wound border were harvested for total RNA extraction. Cytokine mRNA expression was determined using a multi-probe RNase protection assay. Data are presented as means +/- s.e.m. Statistical analysis used the unpaired t-test or Mann-Whitney test where appropriate. Significance was accepted at P < 0.05. Treatment of the burn wound with liposomal IGF-1-cDNA transfer decreased IL-1beta mRNA levels on day 10 after burn trauma from five-fold burn-induced increases compared with sham-treated rats, to near the control values present in unburned skin samples. Similarly, there was an eight-fold increase in TNF-alpha mRNA expression on postburn day 10 that was abrogated by IGF-1 gene therapy. Local IGF-1 gene transfer attenuates the mRNA expression of the inflammatory cytokines IL-1beta and TNF-alpha in the burn wound. This change may improve burn wound healing by decreasing prolonged local inflammation.

Animals↗

PubMed1997

The effects of interleukin-1 and prostaglandin E2 on accumulation of collagen and steady-state levels of pro alpha 1(I) collagen messenger RNA in experimental granulation tissue in rats.

The effects of human interleukin 1 beta (IL-1 beta) and prostaglandin E2 (PGE2) on experimental granulation tissue in rats and on granulation tissue cells in culture were studied. IL-1 beta and PGE2 were injected into subcutaneously implanted sponges during the first 3 days after implantation. The rate of collagen synthesis in fibroblasts was measured as synthesis of protein-bound 3H-hydroxyproline. The steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were estimated by Northern transfer analyses. By 7 days postoperatively IL-1 beta had decreased the hydroxyproline content of granulation tissue. PGE2 decreased non-significantly the amounts of hydroxyproline, but the steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were slightly elevated. In IL-1 beta-treated fibroblast cultures collagen production decreased by 15% and following PGE2 treatment by 34% compared with the controls. The latter effect could be abolished by indomethacin. Indomethacin alone stimulated collagen production by 40%. In vivo IL-1 decreases the formation of normal granulation tissue. This effect may be partly due to stimulation of secretion of PGE2.

Animals↗

PubMed2000

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P.

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.

Base Sequence↗

PubMed2000

A tripeptide 'anticodon' deciphers stop codons in messenger RNA.

The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.

Amino Acid Sequence↗

PubMed1980

Effects of 5-fluorouracil on the formation of modified nucleosides in yeast transfer RNA.

Yeast cells grown in the presence of the drug FUra synthesize RNA in which Urd is partially replaced by FUrd. Transfer RNAs in which 1.5-50% of the Urd has been replaced by FUrd have been isolated and their base compositions measured to determine the effect of FUrd incorporation on posttranscriptional nucleoside modification. This replacement results in an extensive reduction in the amounts of Thd, H56Urd and psird found in mature tRNA. Quantitatively, the reduction of psird greater than or equal to Thd greater than H56Urd. The losses of psird, Thd and H56Urd are greater than can be accounted for by the stoichiometry of FUrd incorporation. The formation of 5-MeCyd is not affected by the drug, whereas the methylated purines show substoichiometric losses in FUrd-containing tRNAs. In Escherichia coli, we have not observed any effects of FUra on the methylated purine content, although the effects on psird, Thd and H56Urd are similar. These findings indicate that (a) in both pro- and eukaryotic systems the enzymes responsible for psird, Thd and H56Urd formation are affected by FUra treatment in a similar manner; (b) prokaryotic purine methylases may be more tolerant of structural aberrations resulting from FUrd incorporation than eukaryotic methylases and (c) different methylases within one system show different sensitivities as shown by those responsible for 1-MeAdo and 5-MeCyd formation.

Base Composition↗

PubMed1976

Anticodon conformation and accessibility in wild-type and suppressor tryptophan tRNA from E. coli.

The association between Trp-tRNA and Pro-tRNA, which have complementary anticodon sequences, has been used as a probe of anticodon conformation. It is unaffected, however, by the base change in the D-stem present in UGA-suppressor Trp-tRNA. This does not support the hypothesis that UGA suppression depends upon a conformational change induced in the anticodon. The stable denatured form of wild-type Trp-tRNA no longer interacts with Pro-tRNA; the structure of the anticodon region must therefore be quite different in the denatured form.

Anticodon↗

PubMed2000

Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers.

The primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNA(B2) to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNA(B2) to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNA(Lys1.2) suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS.

3T3 Cells↗

PubMed1983

Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli.

The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

Amino Acid Sequence↗

PubMed2005

Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library.

The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (approximately 30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis.

Apoptosis↗

PubMed1986

Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis.

Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile). The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete. A majority of the tRNA genes are located in close proximity to one another. A unique feature of the Pro and Ile genes is that their DNA sequence overlap.

Bacteriophages↗

PubMedDate not supplied

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene].

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

Alkaline Phosphatase↗

PubMed1983

Human mitochondrial genome and the evolution of methionine transfer ribonucleic acids.

The recently deciphered sequence of the human mitochondrial genome is analyzed in the light of an archigenetic hypothesis, according to which mitochondria are derived neither from pro- nor eukaryotes but from more primitive organisms. The possibility that animal mitochondria have only one gene both for elongator and initiator methionine tRNA is supported but C-A pair forming cytosine in the anticodon of these tRNAs is considered to be unmodified. The evolution of the gene and of the codon reading pattern of methionine tRNA is discussed.

Animals↗

PubMed2006

Mechanisms regulating immune cell contributions to spiral artery modification -- facts and hypotheses -- a review.

Early during murine, human and porcine pregnancy, endometrium associated with developing placentae is enriched for uterine Natural Killer (uNK) cells. A shared role for uNK cells in each of these species is production of angiogenic growth factors. Many uNK cells are located in close proximity to or structurally integrated within the walls of endometrial vessels. In mice, uNK cells have been found essential for the initiation of pregnancy-associated spiral arterial modification through their production of interferon-gamma. Unique aspects of uNK cell interactions with decidual endothelium are being defined using cell and tissue transfer into pregnant, alymphoid mice, adhesion of viable lymphocytes to frozen uterine tissue sections under shear forces, laser capture microdissection of uNK cells for quantitative RNA analyses and intravital microscopy. These studies indicate that uterine lymphocytes have a fundamental and major importance in promotion of angiogenesis within implantation sites that is regulated by the reproductive hormone cycle. Further, this work suggests that the pro-inflammatory endometrial cytokine response seen when peri-implantation conceptuses arrest has, as its target for destruction, newly developed endometrial vasculature rather than fetal trophoblast.

Animals↗