Conserved features of TBE1 transposons in ciliated protozoa

The complete sequences of four TBE1 transposons from Oxytricha fallax and O. trifallax are presented and analyzed. Although two TBE1s are 98% identical to each other at the nucleotide level, the remaining two TBE1s are only 90% identical both to each other and to the other two. This large evolutionary divergence allows us to identify conserved TBE1 features. TBE1 transposons are 4.1 kbp long and are flanked by 3 bp target-site repeats. The elements consist of 78 bp inverted terminal repeats, of which the 17 terminal base pairs are Oxytricha telomere repeats; a central conserved section of 550 bp that includes a set of nested direct and inverted sequence repeats; and 3 open reading frames conserved for encoded amino acid sequence. The three open reading frames encode a 22 kDa basic protein of unknown function, a 42 kDa ‘D,D35E’ transposase, and a 57 kDa chimeric C ₂ H ₂ zinc finger/protein kinase. The protein kinase domain of the 57 kDa protein is unusual, lacking a conserved ATP-binding motif. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Short Internal Eliminated Sequence with Central Conserved Sequences Interrupting the LA-MSC Gene of the 81 Locus in the Hypotrichous Ciliates Oxytricha fallax and O. trifallax

ABSTRACT IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytricha fallax and O. trifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT… AATG, and thereby resembles a TBE1 transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This implies a excision mechanism for IES-LA that is distinct from those for other known Oxytricha IESs.     

Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.     

Beta-endorphin, adrenocorticotropic hormone, cortisol and catecholamines during aerobic and anaerobic exercise

Twelve non-specifically trained volunteers (aged 26.5 years, SD 3.6) performed exhausting incremental graded exercise (ST) and 1-min anaerobic cycle ergometer exercise (AnT) at 2-h intervals for the purpose of investigating beta-endorphin (beta-E) behaviour dependent on exercise intensity and anaerobic metabolism. In order to determine [beta-E], adrenocorticotropic hormone [ACTH], cortisol [C], adrenaline [A] and noradrenaline [NA] concentrations, venous blood samples were collected prior and subsequent to exercise until the 20th min of the recovery period, as well as in ST before and after exceeding the individual anaerobic threshold (THan,i). Before, during and after ST, lactate concentration, heart rate and perceived degree of exertion were also determined; after AnT maximum lactate concentration was measured. Both types of exercise led to significant increases in [beta-E], [ACTH], [A] and [NA], with levels of [beta-E] and [ACTH] approximately twice as high after ST as after AnT. The [C] increased significantly only after ST. During ST significant changes in [beta-E] and [ACTH] were measured only after exceeding THan,i. At all measuring times before and after ST and AnT both hormones correlated positively. In AnT the increases of [beta-E] and [A] demonstrated a correlation (r = 0.65; P less than 0.05). Both in AnT and ST there was a relationship between the maximum concentrations of beta-E and lactate (r = 0.63 and 0.71; each P less than 0.05). We therefore conclude that physical exercise with increasing or mostly anaerobic components leads to an increase in [beta-E], the extent correlating with the degree of lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Nucleotide sequences of three tRNA genes encoded in Tetrahymena mitochondrial DNA.

Nucleotide sequences of three cloned restriction fragments of Tetrahymena mtDNA which showed hybridization with mitochondrial tRNA have been determined. EcoRI fragment 5 (4.1 kbp) contains the tRNAphe gene sequence with anticodon GAA; Hind III fragment 6 (2.0 kbp) the tRNAhis with anticodon GTG; and EcoRI fragment 7 (1.9 kbp) the tRNAtrp with anticodon TCA. The CCA end is not encoded. All three tRNAs show usual features with common invariant and semi-invariant bases and can be folded into a cloverleaf structure with standard loops and regular base pairs in the stems. However, some minor irregular features are present including several GT pairs and an unmatched TT in the stems, and TCC instead of T psi C. All exhibit high G+C contents (about 50%); in contrast, the flanking regions are extremely A+T rich (about 80%). Several short coding frames can be deduced in these sequences, but their significance is not known.     

Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA

The macronuclear DNA in the ciliated protozoan O. nova consists of integral of 10(7) gene-sized DNA molecules, all of which terminate with 20 bp of C4A4 repeats followed by a 3' (G4T4)2 single-stranded tail. Two immunologically distinct proteins of 55 and 26 kd, which are tenaciously, but noncovalently associated with Oxytricha macronuclear DNA termini, have been purified. These proteins protect DNA termini from degradation by the exonuclease Bal31. They also facilitate retention of natural and synthetic telomeric DNAs onto nitrocellulose. The Oxytricha proteins are not simply C4A4-binding proteins. Rather, their efficient binding requires both the 3' single-stranded (G4T4)2 tail and the adjacent duplex region. Thus, these proteins require both the sequence and the structure of natural DNA termini for efficient binding. As such they represent the first described example of telomeric-specific proteins.     

Muscle metabolic profile and oxygen transport capacity as determinants of aerobic and anaerobic thresholds

Aerobic and anaerobic thresholds determined by different methods in repeated exercise tests were correlated with cardiorespiratory variables and variables of muscle metabolic profile in 33 men aged 20-50 years. Aerobic threshold was determined from blood lactate, ventilation, and respiratory gas exchange by two methods (AerT1 and AerT2) and anaerobic threshold from venous lactate (AnTLa), from ventilation and gas exchange (AnTr) and by using the criterion of 4 mmol.1(-1) of venous lactate (AnT4mmol). In addition to ordinary correlative analyses, applications of LISREL models were used. The 8 explanatory variables chosen for the regression analyses were height, relative heart volume, relative diffusing capacity of the lung, muscle fiber composition, citrate synthase (CS) and succinate dehydrogenase activities, the lactate dehydrogenase--CS ratio, and age. They explained 58% of the variation in AerT1, 73.5% that of AerT2, 71% that of AnTr, 74.5% that of AnTLa, and 67.5% that of AnT4mmol.AerT and AnT alone explained 77% of the variation in each other. Both AerT and AnT were determined mainly by a muscle metabolic profile, with the CS activity of vastus lateralis as the strongest determinant. The factor 'submaximal endurance' which was measured with AerT and AnT seemed to be slightly more closely connected to 'muscle metabolic profile' than was 'maximal aerobic power' (= VO2max), but both also correlated strongly with each other (r = 0.92).     

Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells

Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (approximately 10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed "full-site integration." Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (approximately 0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO4 that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of approximately 10. In a typical reaction at 37 degrees C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to approximately 2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an approximately 70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.     

Metabolic and ventilatory responses to steady state exercise relative to lactate thresholds

The metabolic and ventilatory responses to steady state submaximal exercise on the cycle ergometer were compared at four intensities in 8 healthy subjects. The trials were performed so that, after a 10 min adaptation period, power output was adjusted to maintain steady state VO2 for 30 min at values equivalent to: (1) the aerobic threshold (AeT); (2) between the aerobic and the anaerobic threshold (AeTAnT); (3) the anaerobic threshold (AnT); and (4) between the anaerobic threshold and VO2max (AnTmax). Blood lactate concentration and ventilatory equivalents for O2 and CO2 demonstrated steady state values during the last 20 min of exercise at the AeT, AeAnT and AnT intensities, but increased progressively until fatigue in the AnTmax trial (mean time = 16 min). Serum glycerol levels were significantly higher at 40 min of exercise on the AeAnT and the AnT when compared to AeT, while the respiratory exchange ratios were not significantly different from each other. Thus, metabolic and ventilatory steady state can be maintained during prolonged exercise at intensities up to and including the AnT, and fat continues to be a major fuel source when exercise intensities are increased from the AeT to the AnT in steady state conditions. The blood lactate response to exercise suggests that, for the organism as a whole, anaerobic glycolysis plays a minor role in the energy release system at exercise intensities upt to and including the AnT during steady state conditions.     

Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA.

The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n greater than or equal to 2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.     

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.     

Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules.

We have found abundant telomere-specific terminal transferase activity in crude macronuclear extracts from vegetatively growing cells of the hypotrichous ciliate Oxytricha nova. This activity adds two to seven tandem repeats of the sequence GGGGTTTT (the Oxytricha telomeric repeat) to the 3' end of oligonucleotide primers ending in repeats of G4T4 and always adds the repeats in the proper phase. The activity requires the presence of micromolar amounts of dGTP and dTTP as well as single-stranded oligomer primers ending 3' with repeats of the Oxytricha telomeric sequence. A nuclease activity is present in the extracts which is closely balanced with telomere terminal transferase activity. We propose a simple model for replication of the ends of linear DNA molecules based on the telomere terminal transferase.