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PubMed1992

Haematopoietic cell lines capable of colonizing the thymus following in vivo transfer expressed T-cell receptor gamma-gene immature mRNA.

To clarify the mechanism by which progenitor T (pro-T) cells recognize and enter the thymus, an attempt was made to produce haematopoietic cell lines by the fusion of BALB/c nude mouse bone marrow or foetal liver cells (gestation 14 and 15 days) with AKR thymoma BW5147, thereby immortalizing cells with potency to colonize the thymus, a characteristic of pro-T cells rarely found in adult bone marrow or foetal liver. The hybridomas thus produced were classified according to the phenotype of surface markers, T-cell receptor (TcR) gene configuration and expression. All hybridomas were negative in the surface expression of T-cell markers such as TcR alpha beta, TcR gamma delta, CD3, CD4 and CD8. They had TcR beta-, gamma- and delta-genes, each with a different status with respect to configuration and transcription. Some possessed partially rearranged TcR genes and others expressed immature TcR mRNA. The cell lines were examined for their capacity to colonize the thymus following intravenous injection into recipient mice. It was found that the cells with capacity of colonizing the thymus expressed immature TcR delta mRNA, while the cell lines lacking TcR delta-genes did not home to the thymus. These findings imply that the potency for migrating to thymus is closely associated with the particular stage of prethymic cell differentiation which could be estimated by the analysis of TcR genes, and that some cell lines with the expression of TcR delta-gene mRNA and the ability to colonize the thymus are derived from pro-T cells.

Animals↗

PubMed1985

Inhibition of the aminoacylation of selected tRNA molecules by an estrogen-regulated factor on uterine ribosomes. Regulation of aminoacylation of tRNA by estrogens.

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen-deprived rats. The extracted inhibitor or a native inhibitor-ribosome complex affects the rate of the peptide elongation reaction in a uterine cell-free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl-tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro greater than Val greater than Arg greater than Try greater than Leu greater than Glu greater than Ile greater than Gly greater than His greater than Ser greater than Lys. Inhibition results from a specific dose-dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl-tRNA synthetase. The effect does not result from removal of A-C-C terminal nucleotides from the 3' end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl-tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol-induced inactivation of a ribosome-associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl-tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl-tRNAs to become rate-limiting in the sequential elongation of peptides.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Collagen type I expression in experimental anaplastic thyroid carcinoma: regulation and relevance for tumorigenicity.

Fibrosis in solid malignancies plays a significant role in tumor pathophysiology. Potential mechanisms for collagen type I deposition in anaplastic thyroid carcinoma (ATC) were investigated using 6 characterized ATC cell lines. Three of these cell lines, which produced collagen type I, had, as a group, a poor tumorigenicity when inoculated in athymic mice. This group of cells generated tumors in 4 of 24 injected animals (17%). Pro-alpha 1(I) collagen mRNA-expressing carcinoma and stromal cells were interdispersed in the tumors generated by these ATC cells. By contrast, the 3 noncollagen-producing ATC cell lines were all tumorigenic with a tumor take of 60% in the whole group. In the latter tumors, pro-alpha 1(I) collagen mRNA-expressing cells were confined to the stromal compartment, well delineated from carcinoma cell islets. To study the influence of ATC cells on collagen type I synthesis by fibroblasts, we used AG 1518 diploid human fibroblasts cultured on poly-(2-hydroxyethyl methacrylate) (poly[HEMA])-coated plates. This culture condition allows the study of the effect of collagen mRNA translation in the regulation of collagen type I synthesis. Conditioned media from the 6 ATC cell lines did not influence collagen synthesis. The ATC cell line KAT-4 stimulated fibroblast synthesis of collagen type I when the two cell types were cocultured on poly[HEMA]-coated substrates. Specific inhibitors of PDGF and TGF-beta reduced the KAT 4 carcinoma cell-induced stimulation of collagen type I synthesis. Our data suggest that collagen type I production by carcinoma cells correlates negatively with tumorigenicity and that the formation of a well-defined stroma is of importance for tumor growth. Furthermore, our data suggest that tumor cells are able to stimulate collagen mRNA translation in stromal fibroblasts in direct cell-cell contact by, at least in part, transferring PDGF or TGF-beta.

Animals↗

PubMed2000

Bacterial cell death induced by human pro-apoptotic Bax is blocked by an RNase E mutant that functions in an anti-oxidant pathway.

BACKGROUND: Bax is a member of the Bcl-2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis-inducing activity in the mammalian cells. RESULTS: We isolated a Bax-resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat-resistant, suppressor of Bax-1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N- and C-terminal halves. The introduction of the truncated 5' end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat- and Bax-resistance was not the catalytic site for the endoribonuclease activity of RNase E. CONCLUSIONS: The N-terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti-oxidant pathway and that a mutant RNase E has a novel function as an anti-oxidant.

Apoptosis↗

PubMed1995

tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Binding Sites↗

PubMed2001

Nitric oxide suppresses the expression of Bcl-2 binding protein BNIP3 in hepatocytes.

Nitric oxide (NO) is not only an important signaling molecule, but it also regulates the expression of a number of genes in the liver. We have previously shown that apoptosis in hepatocytes exposed to tumor necrosis factor-alpha and actinomycin D is prevented by NO derived from the inducible nitric-oxide synthase (iNOS), by mechanisms that are both dependent on and independent of modulation of cyclic guanosine monophosphate (cGMP) subsequent to activation of soluble guanylyl cyclase (sGC). We hypothesize that one mechanism by which NO exerts these effects is by regulating the expression of genes involved in apoptosis. We used differential display-polymerase chain reaction to isolate NO-regulated genes in hepatocytes from iNOS knockout mice (to eliminate endogenous inducible NO production). Using this analysis, we identified a NO-suppressed gene fragment homologous with the pro-apoptotic Bcl-2 binding protein BNIP3. Northern analysis confirmed the NO-dependent suppression of BNIP3 in cultured cells. Similarly, the NO donor S-nitroso-N-acetyl-dl-penicillamine (1-1000 microm) down-regulated the expression of BNIP3 in both iNOS knockout and wild-type hepatocytes. This effect of NO was reversed by the sGC inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ),suggesting the involvement of the sGC/cGMP pathway in the modulation of BNIP3 by NO. We propose that suppression of BNIP3 expression is one sGC/cGMP-dependent mechanism by which NO might affect the process of hepatocyte apoptosis.

Adenoviridae↗

PubMed2001

Chinese hamster ovary (CHO) cells may express six beta 4-galactosyltransferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants.

Six beta4-galactosyltransferase (beta4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat(-)2 line of Chinese hamster ovary cells (Gat(-)2 CHO). Two independent mutants termed Pro(-)5Lec20 and Gat(-)2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro(-)5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.

Amino Acid Sequence↗

PubMed1975

Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.

The interaction between Escherichia coli elongation factor-Tu-GTP complex and chemically synthesized 2'(3')-O-aminoacyldinucleoside phosphates with the nucleotide sequence of the 3' terminus of aminoacyl-tRNA (AA-tRNA) has been studied. It was found that C-A-Phe, C-A-Pro, and C-A-Asp interact with EF-Tu-GTP, causing the release of GTP bound to the enzyme. The specificity of this interaction closely resembles that of AA-tRNA since C-A and C-A(Ac-Phe) as well as the corresponding tRNAs are inactive. The 3'-O-aminoacyl derivative C-2'-dA-Phe does not interact with EF-Tu-GTP, whereas the 2'-O-aminoacyl derivative C-3'-dA-Phe is almost as active as the 2'(3')-O-aminoacyl derivative, C-A-Phe. C-A-Phe also interacts with the EF-Tu-GDP complex in a manner similar to its interaction with EF-Tu-GTP. It is concluded that interaction of 2'(3')-O-aminoacyloligonucleotides possessing the sequence of the 3' terminus of AA-tRNA is analogous to the interaction of that terminus with EF-Tu and it is suggested that EF-Tu is specific for 2'-O-AA-tRNA.

Binding Sites↗

PubMed1995

Molecular probing of the secretory pathway in peptide hormone-producing cells.

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.

Amino Acid Sequence↗

PubMed2001

Activation of NF-kB mediates ICAM-1 induction in respiratory cells exposed to an adenovirus-derived vector.

Gene transfer to the respiratory tract by replication-deficient adenoviruses is limited by the induction of inflammatory and immune responses. We previously demonstrated that a E1-E3-deleted recombinant adenovirus carrying the expression cassette for the cystic fibrosis gene (Ad.CFTR) upregulates the expression of the pro-inflammatory intercellular adhesion molecule-1 (ICAM-1) both in vitro and in vivo. In the present work we suggest a role for the nuclear factor-kB (NF-kB) in Ad.CFTR-dependent up-regulation of ICAM-1 in respiratory epithelial A549 cells. Specifically, Ad.CFTR induced translocation of NF-kB into the nucleus and binding to the proximal -228/-218 NF-kB consensus sequence on the ICAM-1 promoter. Ad.CFTR also stimulated a 13-fold increase in NF-kB-dependent expression of the CAT reporter gene under the control of a region of the ICAM-1 promoter, including the proximal NF-kB consensus sequence. The Ad.CFTR-dependent increase of ICAM-1 mRNA was abolished by inhibitors of NF-kB, such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, parthenolide and the synthetic peptide SN50. All these inhibitors abolished both Ad.CFTR-induced NF-kB DNA binding and transactivating activities. These results indicate a critical role of NF-kB in the pro-inflammatory response elicited by replication-deficient adenoviral vectors in respiratory cells.

Acetylcysteine↗

PubMed2004

Hyperglycemia promotes oxidative stress through inhibition of thioredoxin function by thioredoxin-interacting protein.

Increased intracellular reactive oxygen species (ROS) contribute to vascular disease and pro-atherosclerotic effects of diabetes mellitus may be mediated by oxidative stress. Several ROS-scavenging systems tightly control cellular redox balance; however, their role in hyperglycemia-induced oxidative stress is unclear. A ubiquitous antioxidative mechanism for regulating cellular redox balance is thioredoxin, a highly conserved thiol reductase that interacts with an endogenous inhibitor, thioredoxin-interacting protein (Txnip). Here we show that hyperglycemia inhibits thioredoxin ROS-scavenging function through p38 MAPK-mediated induction of Txnip. Overexpression of Txnip increased oxidative stress, while Txnip gene silencing restored thioredoxin activity in hyperglycemia. Diabetic animals exhibited increased vascular expression of Txnip and reduced thioredoxin activity, which normalized with insulin treatment. These results provide evidence for the impairment of a major ROS-scavenging system in hyperglycemia. These studies implicate reduced thioredoxin activity through interaction with Txnip as an important mechanism for vascular oxidative stress in diabetes mellitus.

Animals↗

PubMed1999

Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.

Amino Acid Sequence↗

PubMed2005

Hypothalamic responses to peripheral glucose infusion in food-restricted sheep are influenced by photoperiod.

Nutritional feedback provided by systemic hormones, such as insulin and leptin, influences reproductive neuroendocrine output within the hypothalamus, yet the mechanisms and their interaction with photoperiodic cues remain unresolved in seasonal species. Here, peripheral glucose (G) infusion was used to increase endogenous concentrations of insulin and leptin in food-restricted sheep kept in either long-day (LD) or short-day (SD) photoperiod, and responses were examined in terms of pulsatile luteinising hormone (LH) (gonadotrophin-releasing hormone by inference) output and hypothalamic gene expression for nutritionally sensitive neuropeptides and receptors. We addressed the hypothesis that these hypothalamic responses were correlated and influenced by photoperiod. Oestradiol-implanted, castrated male sheep were kept 16 weeks in SD (8 h light/day) or LD (16 h light/day) and then transferred to the opposite photoperiods for 8 weeks, during which food was restricted to 90% requirement to maintain body weight (maintenance). For the final 6 days, food was reduced to 75% maintenance, and sheep in both photoperiods were infused intravenously with G (60 mM/h) or saline (S) (n = 8/group). G-infused sheep had higher mean plasma concentrations of G, insulin and leptin than S-infused sheep, with no effect of photoperiod. In LD, but not in SD, G infusion increased LH pulse frequency and pulse amplitude. In LD, but not in SD, gene expression in the hypothalamic arcuate nucleus was lower in G- than S-infused sheep for neuropeptide Y (NPY) and agouti-related peptide (AGRP) and was higher in G- than S-infused sheep for pro-opiomelanocortin (POMC). Gene expression for leptin and insulin receptors was not affected by photoperiod or infusion. These results are consistent with the involvement of NPY, AGRP and POMC in mediating the reproductive neuroendocrine response to increased systemic nutritional feedback, and they support the hypothesis that hypothalamic responses to nutritional feedback are influenced by photoperiod in sheep.

Agouti-Related Protein↗

PubMed2003

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Amino Acid Sequence↗

PubMed1993

Mitochondrial DNA sequence evolution in the Arctoidea.

Some taxa in the superfamily Arctoidea, such as the giant panda and the lesser panda, have presented puzzles to taxonomists. In the present study, approximately 397 bases of the cytochrome b gene, 364 bases of the 12S rRNA gene, and 74 bases of the tRNA(Thr) and tRNA(Pro) genes from the giant panda, lesser panda, kinkajou, raccoon, coatimundi, and all species of the Ursidae were sequenced. The high transition/transversion ratios in cytochrome b and RNA genes prior to saturation suggest that the presumed transition bias may represent a trend for some mammalian lineages rather than strictly a primate phenomenon. Transversions in the 12S rRNA gene accumulate in arctoids at about half the rate reported for artiodactyls. Different arctoid lineages evolve at different rates: the kinkajou, a procyonid, evolves the fastest, 1.7-1.9 times faster than the slowest lineage that comprises the spectacled and polar bears. Generation-time effect can only partially explain the different rates of nucleotide substitution in arctoids. Our results based on parsimony analysis show that the giant panda is more closely related to bears than to the lesser panda; the lesser panda is neither closely related to bears nor to the New World procyonids. The kinkajou, raccoon, and coatimundi diverged from each other very early, even though they group together. The polar bear is closely related to the spectacled bear, and they began to diverge from a common mitochondrial ancestor approximately 2 million years ago. Relationships of the remaining five bear species are derived.

Animals↗

PubMed2000

Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch.

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

Alleles↗

PubMed1996

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster.

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region-like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region-like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.

Animals↗

PubMed2003

The effects of dietary n-3 polyunsaturated fatty acids delivered in chylomicron remnants on the transcription of genes regulating synthesis and secretion of very-low-density lipoprotein by the liver: modulation by cellular oxidative state.

The influence of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) (derived from fish or corn oil, respectively) on the expression of mRNA for four genes involved in the regulation of the synthesis, assembly, and secretion of very-low-density lipoprotein (VLDL) in the liver was investigated in normal rat hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). The four genes investigated were those encoding apolipoprotein B (apoB), the microsomal triacylglycerol transfer protein (MTP), and the enzymes acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2), which play a role in the regulation of triacylglycerol and cholesteryl ester synthesis, respectively. mRNA levels for apoB, MTP, and DGAT were unaffected by either fish or corn oil chylomicron remnants, but the amount of ACAT2 mRNA was significantly reduced after incubation of the hepatocytes with fish oil remnants as compared with corn oil remnants or without remnants. These findings indicate that the delivery of dietary n-3 PUFA to hepatocytes in chylomicron remnants downregulates the expression of mRNA for ACAT2, and this may play a role in their inhibition of VLDL secretion. However, when the cells were shifted into a pro-oxidizing or pro-reducing state by pretreatment with CuSO(4) (1 mM) or NAC (5 mM) for 24 hr, levels of mRNA for MTP were increased by about 2- or 4-fold, respectively, by fish oil remnants, whereas corn oil remnants had no significant effect. Fish oil remnants also caused a smaller increase in apoB mRNA in comparison with corn oil remnants in NAC-treated cells (+38%). These changes would be expected to lead to increased VLDL secretion rather than the decrease associated with dietary n-3 PUFA in normal conditions. These findings suggest that relatively minor changes in cellular redox levels can have a major influence on important liver functions such as VLDL synthesis and secretion.

Acetylcysteine↗