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A developmentally regulated gene encodes the dictyostelium homolog of yeast ribosomal protein S4 and mammalian LLRep3 proteins.

We report the sequence and expression of a single-copy gene from Dictyostelium discoideum which encodes the homolog of yeast ribosomal protein S4, a protein located on the small ribosomal subunit and known to play an important role in maintaining translational fidelity. Over a highly conserved central region, the Dictyostelium protein has 78% sequence similarity to the yeast protein and 83% sequence similarity to mammalian S4 protein homologs, the LLRep3 proteins. The Dictyostelium gene encodes a polypeptide 28,717 Da in size and hence this ribosomal protein has been named rp29. The N-terminal sequence of the Dictyostelium rp29 protein is extended by 61 amino acids and 14 amino acids compared to the mammalian and yeast proteins, respectively, and the C-terminus is correspondingly 15 amino acids or 2 amino acids shorter. Although the coding region of the rp29 gene is present on a single exon, a 157bp intron interrupts the 5' untranslated region and unusually contains four direct repeats of the sequence TCAATCT. The gene is expressed maximally during vegetative growth but a second peak of expression also occurs late in development which is restricted to prestalk cells; rp29 is the first Dictyostelium ribosomal protein gene reported which shows prestalk-specific developmental expression. During each round of expression, only a single 0.9kb transcript is produced which is similar in size to the yeast S4 ribosomal protein transcript (0.8kb) but markedly smaller than the mammalian LLRep3 mRNA (1.7kb) due to a much shorter 5' untranslated region.

Amino Acid Sequence↗

Time-dependent changes in receptor/G-protein coupling in rat brain following chronic monoamine transporter blockade.

The potent tropane analog, WF-23 [2beta-propanoyl-3beta-(2-naphthyl) tropane], blocks dopamine, serotonin, and norepinephrine transporters with high affinity in vitro and blocks transporters for at least 2 days following a single in vivo administration. Previous studies demonstrated desensitization of monoamine receptor-coupled G-proteins in brain following chronic treatment of rats with WF-23. The current study sought to determine the time course of this desensitization and the behavioral effects of receptor desensitization. Rats were treated with 1 mg/kg WF-23 and injected i.p. every 48 h for 1 to 21 days. Receptor activation of G-proteins was determined by guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding in brain sections for monoamine receptors, as well as mu opioid receptors as a nonmonoamine receptor control. Chronic treatment with WF-23 produced significant reductions in D(2), 5-hydroxytryptamine 1A, and alpha(2)-adrenergic receptor-stimulated [(35)S]GTPgammaS binding; however, the time course of desensitization varied with different receptors. There was no effect of WF-23 treatment on mu opioid-stimulated [(35)S]GTPgammaS binding at any time point. Consistent with previous studies, there was no effect of WF-23 treatment on D(2) receptor binding, as determined by [(3)H]spiperone autoradiography. Locomotor activity was significantly increased for up to 48 h following acute administration of WF-23, demonstrated by increased photocell beam interruptions. WF-23-induced increases in locomotor activity occurred following repeated administration, as above, for up to 7 days. Following 7 days of treatment, there was a significant decrease in WF-23-increased locomotor activity. This reduction occurred at the same time point as the decrease in D(2) receptor/G-protein coupling, suggesting a role of D(2) desensitization in producing tolerance to WF-23-mediated behavior.

Animals↗

Autoantibodies reacting with heart muscle tissue in coronary heart disease.

By the use of the thrombocyte consumption test (T.C.T.) autoantibodies reacting with heart muscle tissue have been demonstrated in 63.3% of the cases of coronary heart disease accompanied by different grades of insufficiency of the blood supply to the heart muscle. These autoantibodies could be detected two to three weeks after the onset in most patients experiencing a coronary attack for the first time, and persisted for about three to eight weeks following single attacks, whereas following recurrence in patients affected for several months or years the autoantibodies appeared in the serum most frequently within one week, and when the painful attacks were repeated, the T.C.T. remained positive for months, with only short interruptions. In cases of acute myocardial infarction the finding of a positive T.C.T. and its duration did not depend on the severity of the clinical symptoms nor on the measure of the laboratory changes but positive results were obtained more frequently in patients with acute coronary insufficiency (intermediate coronary syndrome) or angina pectoris with intense pain and frequent attacks than in patients in whom the attacks were infrequent producing slight symptoms. There was no evidence that these autoantibodies had any damaging effect.

Antibody Formation↗

Alternation in the fusion complex during constant pacing of sustained ventricular tachycardia.

In a patient with sustained ventricular tachycardia (VT), we observed two different conduction times through the reentry circuit at the critical paced cycle length. The cycle length of the VT was 420 msec and overdrive pacing initially performed at a paced cycle length of 400 msec and repeated at decrements of 10 msec until the VT was interrupted at a paced cycle length of 320 msec. During rapid pacing, constant fusion and progressive fusion were confirmed. The first post-pacing return cycle was identical to each paced cycle length. The conduction time between the stimulus artifact and the orthodromically captured electrogram at the left ventricle was constant at 350 msec in each paced cycle length. However, only at a pacing cycle length of 360 msec two conduction times were alternatively observed, one of 350 msec and the other of 365 msec. When the conduction time changed from 350 msec to 365 msec, morphological alternation both in the surface QRS complex and in the orthodromically captured electrogram was evident. Dual slow pathways or a single slow pathway with plural exits from the reentry circuit is a likely mechanism of the alternation.

Aged↗

The TATA-less promoter of VP1, a plant gene controlling seed germination.

Vp1 is a seed-specific gene involved in the control of dormancy and germination. We here present the complete sequence of the sorghum vp1 promoter/enhancer region highlighting its main features, especially the lack of canonical TATA and CAAT boxes and the presence of elements responsive to abscisic acid and light. The region closest to the start of transcription is highly homologous to the partial proximal sequence reported for the maize vp1 promoter. This region is interrupted by a 57-nt stretch containing 14 CT microsatellite repeats. We observed a poor overall homology to the promoter from abi3 gene, the Arabidopsis counterpart bearing a similar coding sequence. However, there exists a high degree of homology (89%) between a TATA-rich 103-bp stretch of the sorghum vp1 promoter located about 700 nt upstream of the startpoint and miniature inverted transposable elements (MITEs) interspersed within the sorghum seed-specific kafirin cluster. This sorghum MITE-like element displays considerable homology (68%) to the TATA-less promoter from the sorghum NADP-malate dehydrogenase gene and lesser similarity to the Tourist, Pilgrim and Batuta MITEs previously identified within the promoter from the maize Abp1 (auxin-binding protein) gene.

Base Sequence↗

[A measure to continue intra-arterial infusion chemotherapy long-term for locally advanced pancreatic cancer].

For locally advanced non-resectable cancer of the pancreas, we have routinely performed intra-arterial chemotherapy: Each catheter is placed in the splenic artery and gastroduodenal artery during laparotomy, and a mixture of Methotrexate and Angiotensin-II is infused within 30 minutes. This treatment is repeated weekly at our outpatient clinic as long as possible. However, obstruction of the catheter or corresponding artery is the major cause of interruption of treatment. The present paper reports a case in which intra-arterial chemotherapy was possible by repeated catheterization for the catheter obstruction. A 54-year-old woman with non-resectable pancreatic cancer underwent catheter placement during laparotomy, but they became occluded one month later. Another catheter was placed into the common hepatic artery by the Seldinger method. After this catheter was occluded again, another catheter was placed into the inferior pancreaticoduodenal artery via the superior mesenteric artery by the Seldinger method. By repeating this catheter placement, we succeeded in continuing the intra-arterial chemotherapy, and the patient has remained alive (30 postoperative months) without losing her quality of life.

Angiotensin II↗

Using a stopwatch to assess pelvic muscle strength in the urine stream interruption test.

Pelvic muscle strength is important in maintaining urinary continence. The urine stream interruption test provides a simple measure of pelvic muscle strength. This study evaluated the accuracy of the test as adapted for clinical use. Women (n = 75) were tested according to standardized protocol. The test was simultaneously timed using a uroflowmeter (for research purposes) and a stopwatch (a technique more feasible in the practice setting). The stopwatch-timed urine stream interruption test was consistent with the uroflowmeter score (r = 0.90, p < 0.00) and demonstrated adequate repeatability (r = -0.69, p < 0.00). The stopwatch test was related to a digital measure of pelvic muscle strength (r = -0.49, p = 0.00), i.e., women with greater pelvic muscle strength were able to interrupt the stream of urine more quickly. Significantly less involuntary urine loss was seen in women whose stopwatch test score was two seconds or less as compared with those whose scores were greater than two seconds (t = -4.83, p = < 0.00, df = 73). Clinicians can use the urine stream interruption test as a baseline measure and as a tool to assess changes in pelvic muscle strength.

Adolescent↗

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Alleles↗

Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT)n.(GA)n microsatellite embedded within the U2 repeat unit.

The RNU2 locus encoding human U2 small nuclear RNA (snRNA) is organized as a nearly perfect tandem array containing 5 to 22 copies of a 5.8-kb repeat unit. Just downstream of the U2 snRNA gene in each 5.8-kb repeat unit lies a large (CT)n.(GA)n dinucleotide repeat (n approximately equal to 70). This form of genomic organization, in which one repeat is embedded within another, provides an unusual opportunity to study the balance of forces maintaining the homogeneity of both kinds of repeats. Using a combination of field inversion gel electrophoresis and polymerase chain reaction, we have been able to study the CT microsatellites within individual U2 tandem arrays. We find that the CT microsatellites within an RNU2 allele exhibit significant length polymorphism, despite the remarkable homogeneity of the surrounding U2 repeat units. Length polymorphism is due primarily to loss or gain of CT dinucleotide repeats, but other types of deletions, insertions, and substitutions are also frequent. Polymorphism is greatly reduced in regions where pure (CT)n tracts are interrupted by occasional G residues, suggesting that irregularities stabilize both the length and the sequence of the dinucleotide repeat. We further show that the RNU2 loci of other catarrhine primates (gorilla, chimpanzee, orangutan, and baboon) contain orthologous CT microsatellites; these also exhibit length polymorphism, but are highly divergent from each other. Thus, although the CT microsatellite is evolving far more rapidly than the rest of the U2 repeat unit, it has persisted through multiple speciation events spanning > 35 Myr. The persistence of the CT microsatellite, despite polymorphism and rapid evolution, suggests that it might play a functional role in concerted evolution of the RNU2 loci, perhaps as an initiation site for recombination and/or gene conversion.

Animals↗

Metal ions cause the isomerization of certain intramolecular triplexes.

The influence of cations on the capacity of five oligopurine.oligopyrimidine mirror repeat sequences to adopt intramolecular triplexes (the usual H-y3 isomer and/or the rare H-y5 isomer) in recombinant plasmids was investigated. Unexpectedly, the presence of certain metal ions (magnesium, zinc, manganese, and calcium) stabilized the (GAA)4TTCGC(GAA)4 insert in the rare H-y5 when cloned into either of two different sequence backgrounds. Alternatively, either shortening or lengthening the sequence at the central interruption, which becomes the loop of the triplex, led to the formation of the canonical H-y3 under all conditions tested. Similarly, other oligopurine.oligopyrimidine mirror repeat sequences (i.e. (GAA)8 or (GGA)8) formed only the H-y3 under all experimental conditions. All triplexes were stabilized by negative supercoiling at pH 5.0; chemical probe and primer extension analyses served as critical structural tools. Hence, the nature of the central interruption sequence (the loop) of the mirror repeat is important for the stabilization of the H-y5. This region may be important for metal ion binding in the initial stages of triplex formation and thus may play a critical role in determining which isomer is formed. The possible biological role of a DNA sequence adopting three different conformations is discussed.

Cations↗

Correlation of gene and protein structure of rat and human lipocortin I.

Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.

Amino Acid Sequence↗

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori.

Three beta-type early chorion gene copies (6F6.1, 6F6.2, and 6F6.3) are dispersed in the late region of chorion locus Ch1-2. Detailed analysis of the 5'-flanking region and the intron of 6F6.1 shows that they contain sequences that are homologous to Bombyx mori Bm1 repeat elements. Interestingly, the Bm1-type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3'-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6F6.1 5'-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion alpha-gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.

Animals↗

Disruption of the clathrin heavy chain-like gene (CLTCL) associated with features of DGS/VCFS: a balanced (21;22)(p12;q11) translocation.

The smallest region of deletion overlap in the patients we have studied defines a DIGeorge syndrome/velocardiofacial syndrome (DGS/VCFS) minimal critical region (MDGCR) of approximately 250 kb within 22q11. A de novo constitutional balanced translocation has been identified within the MDGCR. The patient has some features which have been reported in individuals with DGS/VCFS, including: facial dysmorphia, mental retardation, long slender digits and genital anomalies. We have cloned the breakpoint of his translocation and shown that it interrupts the clathrin heavy chain-like gene (CLTCL) within the MDGCR. The breakpoint of the translocation partner is in a repeated region telomeric to the rDNA cluster on chromosome 21p. Therefore, it is unlikely that the patient's findings are caused by interruption of sequences on 21p. The chromosome 22 breakpoint disrupts the 3' coding region of the CLTCL gene and leads to a truncated transcript, strongly suggesting a role for this gene in the features found in this patient. Further, the patient's partial DGS/VCFS phenotype suggests that additional features of DGS/VCFS may be attributed to other genes in the MDGCR. Thus, haploinsufficiency for more than one gene in the MDGCR may be etiologic for DGS/VCFS.

Abnormalities, Multiple↗

Junctions of the large single copy region and the inverted repeats in Spinacia oleracea and Nicotiana debneyi chloroplast DNA: sequence of the genes for tRNAHis and the ribosomal proteins S19 and L2.

This work describes the organization, at the nucleotide sequence level, of genes flanking the junctions of the large single copy regions and the inverted repeats of Spinacia oleracea (spinach) and Nicotiana debneyi chloroplast DNAs. In both genomes, trnH1, the gene for tRNA-His(GUG) is located at the extremity of the large single copy region 3' to psbA, the gene for the 35 kd Photosystem 2 protein. Both psbA and trnH1 are transcribed towards the inverted repeat. In spinach, the first 48 codons of rps19, the gene for the chloroplast ribosomal protein S19, lie in the inverted repeat and the last 44 codons lie in the large single copy region at the end opposite to that carrying trnH1. The gene for a protein homologous to the E. coli ribosomal protein L2, rp12, is in the inverted repeat immediately 5' to rps19 and, like rps19, is transcribed towards the large single copy region. In N. debneyi, but not in spinach, rp12 is interrupted by a 666 bp insertion. The gene for tRNA-lle(CAT), trnl1, is located in the inverted repeats of spinach and N. debneyi, 5' to rp12 and is transcribed in the same direction as rp12.

Amino Acid Sequence↗

Exon/intron organization of the gene encoding the mouse epithelin/granulin precursor (acrogranin).

Mouse genomic clones encoding the epithelin/granulin gene and its 5'- and 3'-flanking regions have been isolated and sequenced. This gene was found to be a single-copy gene, and contained 13 exons interrupted by 12 introns. Eight out of the 12 introns are classified as phase 0, and are located within the central part of each of the tandem repeats in the amino acid sequence of the epithelin/granulin precursor. The first intron is unique because of the interruption of the 5'-untranslated region and its fairly large size (approximately 2.4 kbp). Consensus sequences for several of the potential regulatory elements are present in the 5'-flanking sequence, including a common CCAAT sequence.

Amino Acid Sequence↗

Genetic instabilities in (CTG.CAG) repeats occur by recombination.

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.

DNA, Superhelical↗

Triplet repeats and human disease.

In recent years a new mechanism of genetic disease has been discovered in which triplet DNA sequences (trinucleotides) expand either to interrupt or to compromise a gene. So far, ten genetic loci have been identified in which this event takes place. The presence of these expansions, sometimes containing thousands of repeated trinucleotides, provides a clue as to how processing of DNA and RNA may sometimes go awry. How this expansion in a single allele may exert a dominant effect and how the extent of the expansion increases the severity of the disease remain a mystery.

Animals↗

Mechanisms of B cell tolerance: I. Dynamic nature of the induction of hapten-specific unresponsiveness by hapten-conjugated pneumococcal polysaccharide.

Exposure of 2,4-dinitrophenyl (DNP)-hemocyanin-primed spleen cells to DNP-conjugated pneumococcal polysaccharide (DNP-S3) in vivo or in vitro renders such cells unresponsive to DNP-hemocyanin challenge following adoptive transfer. The unresponsiveness is hapten-specific, independent of the presence of T cells and adherent cells, and not due to either toxic effect of S3 or carry-over of tolerogenic amounts of cell-bound DNP-S3, and thus presumably represents a model of B cell tolerance. The degree of suppression induced depends upon the dose of the tolerogen and most strikingly on the duration of exposure of cells to the tolerogen. Thus 2 to 6 h exposure to DNP-S3 has an insignificant effect on anti-DNP responses, while 24 and 48 h exposures in vitro and in vivo are highly suppressive. Such a dynamic process of tolerance induction suggests a "multiple hit" phenomenon, implying the generation of suppressive signals by repeated cycles of tolerogen-receptor interactions. The process can be interrupted by removal of extracellular DNP-S3, although this does not reverse the unresponsiveness that has already been induced.

Animals↗