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PubMed1993

Characterization of the terminal inverted repeats and their neighboring tandem repeats in the Chlorella CVK1 virus genome.

A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was constructed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophoresis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approximately 1.0 kb. Adjacent to the ITR, unique sequence elements of 10 to 20 bp were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally generated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.

Base Sequence↗

PubMed1988

Formation of deletion in Escherichia coli between direct repeats located in the long inverted repeats of a cellular slime mold plasmid: participation of DNA gyrase.

We constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp. strain GA11), and using pAG60 as cloning vector. We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells. However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene. These results suggest that E. coli DNA gyrase is involved in the mechanisms of the deletion formation. It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region. Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats [21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT], located near the distal ends of the inverted repeats, preserving one copy of the repeats. These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions.

Base Sequence↗

PubMed1997

A novel repeat sequence (CKRS-1) containing a tandemly repeated sub-element (kre) accounts for differences between Candida krusei strains fingerprinted with the probe CkF1,2.

CkF1,2 has been reported as an effective DNA fingerprinting probe of Candida krusei. It is composed of two genomic EcoRI-restriction fragments, F1 and F2, which are approximately 5.4 and 5.2 kb, respectively. Sequence analysis of F1 reveals that it is 5261 bp-long, has a GC content of 42.2 mol%, and originates from the intergenic region of the ribosomal RNA cistrons (IGR). F1 comprises 488 bp of the 3' end of a 25s rRNA gene, a non-transcribed spacer region 1 (NTS1), a 5s gene (121 bp), and a major portion of the non-transcribed spacer region 2 (NTS2). A 1256 bp-long repeated sequence, CKRS-1, with a GC content of 35 mol%, has been identified in NTS2. CKRS-1 contains eight tandemly repeated sub-elements, kre-0 to kre-7. The first two, kre-0 and kre-1, are 164 bp-long, the next five sub-elements, kre-2 to kre-6, are 165 bp-long, and the last element, kre-7, is 103 bp-long. The eight sub-elements share nucleotide-sequence homologies between 66 to 100%, with kre-2, kre-3 and kre-4 identical, and kre-0 the most divergent. Shorter repeated sequences were also identified in three regions of F1, which were named domains "a", "b" and "c". Restriction mapping, cross hybridization, and direct comparison of sequences show that F1 and F2 are polymophic forms of the IGR and their size difference is due both to the number of kre sub-elements in CKRS-1 and to a 24-bp deletion in domain "b". While F1 contains eight kre sub-elements, F2 contains seven. In C. krusei strain K31, four polymorphic forms of CKRS-1 have been identified containing five, six, seven and eight kre sub-elements. CKRS-1 is dispersed on three of the chromosomes of highest molecular weights separated by transverse alternating-field electrophoresis. CKRS-1 does not hybridize significantly to any transcription product. Polymorphisms in single DNA fingerprints and differences between the DNA fingerprints of strains of C. krusei based upon CkF1,2 hybridization patterns therefore appear to be based, at least in part, on the variable number of tandemly repeated kre sub-elements in CKRS-1.

Base Sequence↗

PubMed1997

The highly rearranged chloroplast genome of Trachelium caeruleum (Campanulaceae): multiple inversions, inverted repeat expansion and contraction, transposition, insertions/deletions, and several repeat families.

Comprehensive gene mapping reveals that the chloroplast genome of Trachelium caeruleum is highly rearranged relative to those of other land plants. Evolutionary scenarios that consist of seven to ten inversions, one or two transpositions, both expansion and contraction of the typically size-conserved inverted repeat, a presumed gene loss, deletions within two large open reading frames and several insertions, are sufficient to derive the Trachelium arrangement from the ancestral angiosperm chloroplast DNA arrangement. Two of the rearrangements disrupt transcriptional units that are otherwise conserved among land plants. At least five families of small dispersed repeats exist in the Trachelium chloroplast genome. Most of the repeats are associated with inversion endpoints and may have facilitated inversions through recombination across homologous repeats.

Biological Evolution↗

PubMed1989

Linkage in human heterochromatin between highly divergent Sau3A repeats and a new family of repeated DNA sequences (HaeIII family).

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp] were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a "new" family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.

Base Sequence↗

PubMed1983

A mouse dispersed repeat sequence showing remarkable similarities to the long terminal repeats of retroviruses.

A mouse 1.3-kb EcoRI dispersed repeat sequence that shows homology by hybridisation to the long terminal repeat of intracisternal A-particle genes has been partially sequenced. One of the established sequence regions shows three major features reminiscent of the long terminal repeats of retroviruses. Firstly, a TATA box precedes a putative capping site (G) by 23 bp. Secondly, the TATA box is itself preceded by the sequence CCTAT. Thirdly, following the capping site, the signal sequence AATAA precedes by 20 bp a possible polyadenylation site (CA). Such a retroviral-like sequence may have been responsible for the initial amplification and dispersion of the mouse repeat family.

Animals↗

PubMed1997

Follicular dynamics, repeatability and predictability of follicular recruitment in cows undergoing repeated follicular puncture.

The aim of our study was to characterize follicular recruitment and growth in response to the repeated removal of follicles. All tertiary follicles (> 2 mm of diameter) in the ovaries of 10 non-lactating Holstein Friesian cows were punctured at midcycle (Day 0) by means of an ultrasound-guided needle. Puncture sessions were scheduled twice weekly at 3- or 4-d intervals over 3 mo. In the middle of the experiment, i.e., Week 7, the effects of 2- and 5-d intervals between follicular punctures were tested and compared with the previous 3- and 4-d intervals. After this period, 6 animals were slaughtered to study the effect of puncturing on gross ovarian morphology. The protocol of puncturing follicles with 3- to 4-d intervals was continued for an additional 3 mo in the remaining 4 animals. Twice-weekly puncturing of all tertiary follicles abolished estrous cycles and lead to an increase in follicular wave frequency without apparent negative effects on either the reproductive tract or ovaries. After puncture the new follicular wave attained full numerical development within 3 d. Two-day intervals resulted in a lower number of follicles than the 3-d interval (11.0 -/+ 3.8 vs 15.4 -/+ 6.1; P < 0.05). In contrast 4- and 5-d intervals between puncture resulted in an increase in follicle size when compared with that of the 3-d interval. The mean+SD number of recruited follicles varied between animals ranging between 78 +/- 2.5 to 19.2 -/+ 6.0. The mean number of follicles recruited increased from the first month (March) to the third month (May) of sampling (11.8 +/- 4.7 vs 16.4 +/- 6.5; P < 0.01), and then decreased between the third (May) and the sixth (August) month of sampling (21.5 +/- 4.7 vs 16.8 +/- 5.0; P < 0.01). During the experiment, the number of recruited follicles varied cyclically, with waves having a length of 6 puncture session (PS) or 3 wk (i.e., the mean length the bovine estrous cycle). Follicular recruitment after repeated ovum pick-up showed a high repeatability (r = 0.576) A model was also developed showing good predictability of the potential of animals to recruit follicles on the basis of the first 4 to 6 PS. Our results showed that despite large variation in follicular recruitment between animals, the high repeatability and good predictability of follicle recruitment demonstrates the possibility of characterizing animals on their potential for follicle growth.

Journal Article↗

PubMed1987

Effect of repeated episodes of drug-induced ventricular dyskinesia on subsequent regional function in the dog: comparison with myocardial stunning produced by repeated coronary occlusions.

Stunned myocardium can be produced by repeated short episodes of ischemia. Histochemical and ultrastructural abnormalities such as sarcomere lengthening and myofiber thinning have been noted in myocardium soon after the onset of ischemia and have been attributed to the mechanical stretching that occurs during ventricular systole. To test whether mechanical forces alone could produce the residual dysfunction seen in stunned myocardium, regional dyskinesia was produced in open chest dogs by six repeated intracoronary infusions of either potassium chloride, 0.2 mEq/min for 2.5 minutes, or lidocaine, a 10 mg bolus followed by 1 to 3 mg/min for 5 minutes. These dogs were matched with dogs that had six repeated coronary occlusions of 2.5 and 5 minutes' duration, respectively. Regional function was analyzed using fractional systolic shortening and the load-independent end-systolic pressure-length relation. Both potassium chloride and lidocaine produced regional dyskinesia that was similar to the dyskinesia produced by coronary occlusion. Although regional ventricular function after repeated coronary occlusions remained significantly reduced, function returned completely to normal within 5 minutes after the last drug-induced dyskinesia. In conclusion, regional dysfunction produced by potassium chloride and lidocaine does not produce residual dysfunction despite mechanical forces during systole similar to those seen during coronary occlusion.

Animals↗

PubMed2001

Spontaneous and ultraviolet light-induced direct repeat recombination in mammalian cells frequently results in repeat deletion.

Recombination is enhanced by transcription and by DNA damage caused by ultraviolet light (UV). Recombination between direct repeats can occur by gene conversion without an associated crossover, which maintains the gross repeat structure. There are several possible mechanisms that delete one repeat and the intervening sequences (gene conversion associated with a crossover, unequal sister chromatid exchange, and single-strand annealing). We examined transcription-enhanced spontaneous recombination, and UV-induced recombination between neomycin (neo) direct repeats. One neo gene was driven by the inducible MMTV promoter. Multiple (silent) markers in the second neo gene were used to map conversion tracts. These markers are thought to inhibit spontaneous recombination, and our data suggest that this inhibition is partially overcome by high level transcription. Recombination was stimulated by transcription and by UV doses of 6-12J/m(2), but not by 18J/m(2). About 70% of spontaneous and UV-induced products were deletions. In contrast, only 3% of DSB-induced products were deletions. We propose that these product spectra differ because spontaneous and UV-induced recombination is replication-dependent, whereas DSB-induced recombination is replication-independent.

Animals↗

PubMed1998

A comparison of repeated high doses and repeated standard doses of epinephrine for cardiac arrest outside the hospital. European Epinephrine Study Group.

BACKGROUND: Clinical trials have not shown a benefit of high doses of epinephrine in the management of cardiac arrest. We conducted a prospective, multicenter, randomized study comparing repeated high doses of epinephrine with repeated standard doses in cases of out-of-hospital cardiac arrest. METHODS: Adult patients who had cardiac arrest outside the hospital were enrolled if the cardiac rhythm continued to be ventricular fibrillation despite the administration of external electrical shocks, or if they had asystole or pulseless electrical activity at the time epinephrine was administered. We randomly assigned 3327 patients to receive up to 15 high doses (5 mg each) or standard doses (1 mg each) of epinephrine according to the current protocol for advanced cardiac life support. RESULTS: In the high-dose group, 40.4 percent of 1677 patients had a return of spontaneous circulation, as compared with 36.4 percent of 1650 patients in the standard-dose group (P=0.02); 26.5 percent of the patients in the high-dose group and 23.6 percent of those in the standard-dose group survived to be admitted to the hospital (P=0.05); 2.3 percent of the patients in the high-dose group and 2.8 percent in the standard-dose group survived to be discharged from the hospital (P=0.34). There was no significant difference in neurologic status according to treatment among those discharged. High-dose epinephrine improved the rate of successful resuscitation in patients with asystole, but not in those with ventricular fibrillation. CONCLUSIONS: In our study, long-term survival after cardiac arrest outside the hospital was no better with repeated high doses of epinephrine than with repeated standard doses.

Adrenergic Agonists↗

PubMed2004

C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats.

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Amino Acid Sequence↗

PubMed1987

Human C3b/C4b receptor (CR1). Demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristics of C3/C4 binding proteins.

10 overlapping CR1 cDNA clones that span 5.5 kb were isolated from a tonsillar library and sequenced in whole or in part. A single long open reading frame beginning at the 5' end of the clones and extending 4.7 kb downstream to a stop codon was identified. This sequence represents approximately 80% of the estimated 6 kb of coding sequence for the F allotype of CR1. Three tandem, direct, long homologous repeats (LHRs) of 450 amino acids were identified. Analysis of the sequences of tryptic peptides provided evidence for a fourth LHR in the F allotype of CR1. Amino acid identity between the LHRs ranged from 70% between the first and third repeats to 99% between the NH2-terminal 250 amino acids of the first and second repeats. Each LHR comprises seven short consensus repeats (SCRs) of 60-70 amino acids that resemble the SCRs of other C3/C4 binding proteins, such as complement receptor type 2, factors B and H, C4 binding protein, and C2. Two additional SCRs join the LHRs to a single membrane-spanning domain of 25 amino acids; thus, the F allotype of CR1 probably contains at least 30 SCRs, 23 of which have been sequenced. Each SCR is predicted to form a triple loop structure in which the four conserved half-cystines form disulfide linkages. The linear alignment of 30 SCRs as a semi-rigid structure would extend 1,140A from the plasma membrane and might facilitate the interaction of CR1 with C3b and C4b located within the interstices of immune complexes and microbial cell walls. The COOH-terminal cytoplasmic domain of 43 residues contains a six-amino-acid sequence that is homologous to the sequence in the epidermal growth factor receptor that is phosphorylated by protein kinase C.

Amino Acid Sequence↗

PubMed1982

Spacer size heterogeneity in ribosomal DNA of Chironomus thummi is due to a 120 bp repeat homologous to a predominantly centromeric repeated sequence.

The rDNA of Ch. tepperi is homogeneous in structure with a repeat size of 8.4 kb. This size seems to be typical for the basic repeat unit in Chironomus species. Ch. th. piger rDNA cistrons are slightly increased in length (9.0 kb). In the non-transcribed spacer (NTS) an appr. 0.18 kb segment is additionally present in about 50% of the repeats. Ch. th. thumni DNA contains largely heterogeneous rDNA repeats, mainly between 10 and 16 kb. The heterogeneity is due to varying numbers of 120 bp elements present in the NTS. The different spacer size classes are not randomly distributed. The short repetitive 120 bp elements (Cla I elements) hybridize in situ with the nucleolus and with centromere regions. The Cla I elements are regularly present in the thummi NTS, but are absent in the piger NTS. Only very few piger rDNA cistrons may contain Cla I elements.

Animals↗

PubMed2000

Complete deletion of yeast chromosomal rDNA repeats and integration of a new rDNA repeat: use of rDNA deletion strains for functional analysis of rDNA promoter elements in vivo.

Strains of Saccharomyces cerevisiae were constructed in which chromosomal rDNA repeats are completely deleted and their growth is supported by a plasmid carrying a single rDNA repeat, either a plasmid carrying the 35S rRNA gene transcribed from the native promoter by RNA polymerase I or a plasmid carrying the 35S rRNA gene fused to the GAL7 promoter for transcription by RNA polymerase II. This system has made it possible to assess the expression of rDNA by measuring the ability of synthesized rRNA to support cell growth as well as by measuring the actual rRNA synthesized rather than by the use of reporter mini-rDNA genes. Using this system, deletion analysis of the rDNA promoter confirmed the presence of two elements, the upstream element and the core promoter, and showed that basal transcription from the core promoter, if it takes place in vivo as was observed in vitro, is not sufficient to allow cell growth. We have also succeeded in integration of a rDNA repeat and its copy number expansion at the original chromosomal locus, which will allow future mutational analysis not only of rRNA but also other DNA elements involved in rRNA transcription, rDNA replication and recombination within a repeated rDNA structure.

Chromosomes, Fungal↗

PubMed1977

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.

Animals↗

PubMed1997

A retrotransposon of the non-long terminal repeat class from the human blood fluke Schistosoma mansoni. Similarities to the chicken-repeat-1-like elements of vertebrates.

The genomes of representative species of fishes, amphibians, and reptiles contain non-long-terminal-repeat (non-LTR) retrotransposons showing strong sequence identity to the chicken repeat 1 (CR1) non-LTR retrotransposon from birds. These nonavian retroelements have been termed CR1-like elements. We have isolated sequences of a non-LTR retrotransposon from the human blood fluke Schistosoma mansoni. These schistosome sequences, which we have termed the SR1 family of non-LTR retrotransposons, contain regions of deduced amino acids characteristic of the CR1-like elements. SR1 elements possess atypical 3' termini consisting of the tandem repeat (AACCATTTG)2 which are similar in structure to the imperfect tandem repeat of the 3' termini of CR1. There are at least 200 copies of SR1 interspersed through the genome of S. mansoni. The structural and amino acid sequence similarities of SR1 with members of the CR1-like elements suggest that the SR1 family belongs to the CR1-like category of non-LTR retrotransposons. Although other non-LTR retrotransposons have been described in invertebrates, this is the first CR1-like element reported from a nonvertebrate taxon, suggesting that the phylogenetic distribution of CR1-like retrotransposons is not restricted to vertebrates.

Amino Acid Sequence↗

PubMed1997

Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

Amino Acid Sequence↗

PubMed1999

Irritancy ranking of anionic detergents using one-time occlusive, repeated occlusive and repeated open tests.

Discrepancies between the one-time patch test and the wash test regarding the ranking of irritancy of detergents have been found in the literature. The aim of the present study was to investigate the concordance of irritancy rank order of 4 anionic detergents tested by 3 different exposure methods, namely one-time occlusive, repeated short-time occlusive and repeated short-time open tests. These detergents were sodium cocoyl isethionate (ISE), sodium lauryl sulfate (SLS), soap and disodium lauryl 3-ethoxysulfosuccinate (SUC). The reactions were evaluated by visual scoring and by transepidermal water loss (TEWL) measurement. When scored visually, the rank order in the one-time test was: SOAP > or = SLS > or = ISE > SUC. The other test methods yielded a different order: SLS > ISE > or = SOAP > SUC. A similar rank order was obtained with TEWL measurement for all exposure methods. Generally, the concordance among the different exposure methods was high when evaluated by TEWL. The concordance was lower when evaluation was performed by visual scoring. The present study demonstrates that the choice of exposure model and evaluation method may be important variables influencing the outcome of irritancy testing. It is proposed that the repeated open test is the best way to simulate most in-use situations where the uncovered skin is exposed to detergents. The repeated occlusive test or the one-time patch test may be better to simulate situations in which the skin is occluded after irritation by detergents.

Adolescent↗