Repeating units of Xenopus laevis oocyte-type 5S DNA are heterogeneous in length

Princips of computer tomography is demonstrated and the absorption coefficients of ocular and orbital tissue are calculated. The advantages and disadvantages of this procedure are discussed. 39 patients with ocular and orbital diseases were examined by computer tomography. The main indication was tumors, including metastases and diseases arising from ethmoidal cells. Vascular anomalies and tumors were better demonstrated by angiography. Dislocated lens, enlarged tumors of the choroid and prostheses (cerclage and orbital implants) are demonstrable. In contrast to echography, the computer tomography gives certainly better results in the posterior part of the orbit. Technical improvement of the apparatus could lead to a wider use in ophthalmology.     

Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis

LY171883, a peroxisome proliferator and leukotriene D4-antagonist, induced a statistically significant increase in the number of hepatic lesions in B6C3F1 female mice in a 2 year oncogenicity study at dietary doses of 0.0225% and 0.075%. The mutation frequency and spectrum of the 61st codon of H-ras was determined for 64 independent, archived lesions from the LY171883 2 year oncogenicity study using the polymerase chain reaction (PCR), allele specific oligo hybridization (ASO) and DNA sequencing. Results showed 41 (64%) of these lesions had mutations at the 61st codon (16/21 hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26 focal hepatocellular hyperplasias and 2/7 focal hepatocellular atypia). These mutations consisted of 18 C-A transversions, 16 A-G transitions and seven A-T transversions. Compared to the mutation frequency for spontaneously occurring archival B6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions (64%) was significantly higher (P < 0.01). The frequencies of H-ras 61st codon mutations among the LY171883 lesion types (hepatocellular carcinomas 76%, hepatocellular adenomas 40%, focal hepatocellular hyperplasias 73% and hepatocellular atypia 29%) were also significantly different (P = 0.035). In contrast, spontaneous lesions showed no statistical difference in the frequencies of mutation among lesion types (P > 0.5). The mutation spectrum of the LY171883 lesions was not significantly different from the spontaneous spectra. It may be concluded that based on the similarity in mutation spectrum and the increase in mutation frequency, LY171883 may selectively promote spontaneous hepatic lesions containing H-ras 61st codon mutations. In addition, the difference in mutation frequency among lesion types does not support a linear progression of all LY171883 lesions through focal atypia, focal hepatocellular hyperplasias, hepatocellular adenomas and hepatocellular carcinomas.     

An inhibitor of DNA topoisomerase I from Xenopus laevis ovaries

A novel, heat-resistant and Pronase-sensitive, inhibitor of eukaryotic DNA topoisomerase I has been purified from Xenopus laevis ovaries. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the most purified fraction revealed three bands with apparent molecular masses of 25, 28.5, and 33.5 kDa. The 25- and 33.5-kDa peptides recovered from an SDS-PAGE gel inhibited X. laevis DNA topoisomerase I. The purified inhibitor was specific to DNA topoisomerase I and did not inhibit other DNA enzymes tested. The inhibitor blocked the catalytic activity of DNA topoisomerase I by interacting with the enzyme, rather than by competing for binding sites on substrate DNA. Binding of DNA topoisomerase I to substrate DNA was blocked by the inhibitor, as was the cleavage reaction catalyzed by DNA topoisomerase I. Inhibition of DNA topoisomerase I was relieved by divalent cations Ca2+, Mg2+, or Mn2+.     

Characterization of FEN-1 from Xenopus laevis. cDNA cloning and role in DNA metabolism

cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --> 3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.     

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains

66 inpatients who met the DSM-III-R and ICD-10 criteria for paranoid schizophrenia-active phase were assessed using the SAPS and CDS in order to establish interrelations between depressive and positive symptomatology. The results were then analyzed in reference do particular items of the scale CDS and the three stages of examinations. Numerous positive correlations were found between depressive symptoms and the symptoms from the subscale of Delusions and Hallucinations SAPS (Delusions of Sin or Guilt, of Mind Reading, of Being Controlled, of Reference, Somatic Delusions and Hallucinations) and negative correlations with the symptoms from the subscale of Positive Formal Thought Disorder.     

Purification and partial characterization of (6-4) photoproduct DNA photolyase from Xenopus laevis

Liposarcomas of the head and neck are exceedingly rare, and fewer than 90 cases have been reported in the literature. Liposarcoma of the oral cavity is an even less common entity, and to our knowledge only nine cases have been reported to date. We report the clinical and pathologic findings of a case of well-differentiated liposarcoma of the base of tongue and tonsillar fossa. The patient is a 76-year-old white man with a long-standing history of a mass in the oral cavity and hypopharynx. The mass had been resected several times over the span of 23 years, and diagnoses of lipoma, neurofibroma, mesenchymoma, and angiofibrolipoma have been rendered on different occasions. At the last admission, a polypoid mass of the left tonsillar fossa and base of tongue was resected. The tumor was multinodular and measured 2.5 cm in greatest diameter. Histologically the tumor was ill-defined with infiltrating borders and was composed predominantly of mature adipose tissue with occasional lipoblasts. A small proportion of the tumor consisted of clusters of spindle cells and pleomorphic lipoblasts. Mitotic activity was not seen. The pleomorphic cells were positive for S100 protein and negative for muscle-specific markers. Ultrastructural analysis confirmed the nature of the lipoblasts. Our case depicts the typical natural history and histologic features of liposarcoma of the oral cavity. This tumor is usually well differentiated and has a high recurrence rate and almost no tendency for metastasis. Based on our case and review of the literature, it appears that well-differentiated liposarcoma of the oral cavity can occasionally be underdiagnosed because of the low mitotic activity and long latent period between the original diagnosis and first recurrence.     

A novel nuclease activity from Xenopus laevis releases short oligomers from 5'-ends of double- and single-stranded DNA

BACKGROUND: Double-strand breaks in chromosomal DNA of eucaryotic cells are assumed to be repaired by mechanisms of illegitimate recombination capable of direct rejoining of the broken ends. Cell-free extracts of Xenopus laevis eggs efficiently perform these end joining reactions with any pair of noncomplementary DNA termini whose single-stranded 5'- or 3'-overhangs do not exceed a length of approximately 10 nt. RESULTS: Using hairpin-shaped oligonucleotides that allow the construction of double-strand break termini with 5'- or 3'-overhangs of defined length and sequence we show that 5'-overhangs of more than 9-10 nt are exonucleolytically resected in the extract to produce shorter 5'-overhangs that can be metabolized in the end joining reaction. 5'-recessed ends in double-stranded DNA with 3'-overhangs of more than 2nt as well as the 5'-ends of single-stranded DNA also serve as substrates for the exonuclease activity. In all cases, oligomers of about 10 nt are released from the 5'-ends. CONCLUSIONS: We describe here a novel 5'-exonuclease activity present in eggs from Xenopus laevis that reproducibly removes decameric oligonucleotides from 5'-ends of double- and single-stranded DNA. A possible function of this unusual activity is discussed in the context of homologous and illegitimate genetic recombination processes.     

Purification and some properties of ribonuclease from Xenopus laevis eggs

A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80 degrees C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y: Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.     

DNA-dependent RNA polymerase C from Xenopus laevis ovaries. Ability to transcribe intact double-stranded DNA

When human erythrocyte acetylcholinesterase (AChE) is inhibited by succinyldicholine (SuCh) at low ionic strength, mu = 0.03, two apparent affinity constants for the inhibitor can be distinguished, KI(1) = 5.4 X 10-5 M and KI(2) = 1.3 X 10-5 M. Increasing the ionic strength to mu = 0.58 increases KI(1) to 1.1 X 10-4 M, but abolishes the contribution of KI(2) completely. It is suggested that a similar process may underlie the apparently continuous variation in affinity constant with ionic strength which has been reported for many bifunctional cationic inhibitors. Since KI(2) is in a concentration range commonly attained therapeutically, some of the clinical manifestations of succinyldicholine administration, e.g., muscarinic effects, may be explained by its anticholinesterase action.     

Progesterone-induced DNA polymerase activity in full-grown oocytes of Xenopus laevis (1)

DNA polymerase activity increases in full-grown oocytes of Xenopus laevis during in vitro progesterone-induced maturation. This increase is inhibited by cycloheximide. The presence of the oocyte's nucleus (germinal vesicle) seems essential for the induction of this increase: in previously enucleated oocytes, the level of DNA polymerase activity does not change during progesterone treatment. Furthermore, a new form of DNA polymerase is detectable by DEAE chromatography in in vitro matured oocytes.     

Development of the gut in Xenopus laevis

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.     

Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates

The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety.     

Extraordinary ribosomal spacer length heterogeneity in a neotyphodium endophyte hybrid: implications for concerted evolution

An extraordinary level of length heterogeneity was found in the ribosomal DNA (rDNA) of an asexual hybrid Neotyphodium grass endophyte, isolate Lp1. This hybrid Neotyphodium endophyte is an interspecific hybrid between two grass endophytes, Neotyphodium lolii, and a sexual form, Epichl?e typhina, and the length heterogeneity was not found in either of these progenitor species. The length heterogeneity in the hybrid is localized to the intergenic spacer (IGS) and is the result of copy-number variation of a tandemly repeated subrepeat class within the IGS, the 111-/119-bp subrepeats. Copy number variation of this subrepeat class appears to be a consequence of mitotic unequal crossing over that occurs between these subrepeats. This implies that unequal crossing over plays a role in the concerted evolution of the whole rDNA. Changes in the pattern of IGS length variants occurred in just two rounds of single-spore purification. Analysis of the IGS length heterogeneity revealed features that are unexpected in a simple model of unequal crossing over. Potential refinements of the molecular details of unequal crossing over are presented, and we also discuss evidence for a combination of homogenization mechanisms that drive the concerted evolution of the Lp1 rDNA.     

Solubilization and biochemical characterization of the melatonin deacetylase from Xenopus laevis retina

Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N-acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.     

Complete replication of human sperm genome in egg extracts from Xenopus laevis

Significant amounts of D-aspartate (Asp) are found in mammalian tissues and D-Asp is presumed to play some significant, but as yet undefined physiological role. However, it is not known whether D-Asp is synthesized in mammals. In this study, we addressed this issue in cultured rat pinealocytes, parenchymal cells of the pineal gland, which contain significant amounts of D-Asp. Biosynthesis of D-Asp was found to be minimal to non-existent in cultured rat pinealocytes. We then investigated the mechanism of uptake of D-Asp into these cells and its consequent effect on cell function. D-Asp was efficiently taken up into cells, in a time- and dose-dependent manner. Interestingly, the L-Asp levels in the cells and media decreased concomitantly with the uptake of D-Asp. This decrease was not due to D-Asp cytotoxicity, since the cellular levels of othernted. D-Serine and D-alanine were not taken up efficiently into the cells and the cellular levels of L-serine and L-alanine were unchanged. Also, immunocytochemical staining with anti-D-Asp antibody showed that D-Asp, which had been taken up into the cells, was dispersed throughout the cytoplasm. In response to norepinephrine stimulation, pinealocytes, which had been pretreated with D-Asp released D-Asp as well as L-Asp. In these cells, norepinephrine-induced secretion of melatonin, a pineal hormone, was suppressed. The mechanism of this suppression is discussed here.