Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element.

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 degrees C, but not at 25 degrees C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.

Intestinal Behçet's disease associated with myelodysplastic syndrome with chromosomal trisomy 8--a report of two cases and a review of the literature.

Two cases of intestinal Behçet's disease, which developed in the state of myelodysplastic syndrome with trisomy 8, are presented. Both cases are included in the incomplete type of Behçet's disease, with recurrent aphthous stomatitis, skin lesions, genital ulcers or vascular involvement and punched-out ulcers in the cecum, without ocular involvement. The chromosomal analyses revealed chromosomal abnormalities, including trisomy 8, in both cases. Chromosomal trisomy 8 was shown in all 6 cases with the intestinal Behçet's disease associated with myelodysplastic syndrome reported previously, including our patients. Their histories indicated that myelodysplastic syndrome might have started before the development of intestinal Beçet's disease. Theses findings suggested that chromosomal trisomy 8 might play an important role in the pathogenesis, at least in some groups, of intestinal Behçet's disease.

Protein kinase C inhibitor, H-7 suppresses the growth activity of hepatoma-derived growth factor.

BACKGROUND/AIMS: A well-differentiated hepatocellular carcinoma-derived cell line, HuH-7 proliferates autonomously in serum-free medium. Human hepatoma-derived growth factor, which was purified from the conditioned medium of HuH-7 cells, stimulates the growth of HuH-7 cells by an autocrine fashion, and fibroblasts and endothelial cells by a paracrine fashion. We investigated the role of protein kinase C in the proliferation of HuH-7 cells and the growth activity of hepatoma-derived growth factor. METHODOLOGY: The effects of a selective protein kinase C inhibitor, H-7 on the proliferation of HuH-7 and 3T3 fibroblasts stimulated by hepatoma-derived growth factor were examined by DNA synthesis and cell growth assay. RESULTS: H-7 suppressed the growth of HuH-7 cells. The ID50 of H-7 on the growth of HuH-7 cells was about 25 microM, and the growth of HuH-7 cells was almost completely inhibited by not less than 50 microM of H-7. H-7 inhibited the growth activity of hepatoma-derived growth factor for Swiss 3T3 fibroblasts. The ID50 of H-7 on the activity of hepatoma-derived growth factor for 3T3 fibroblasts was about 25 microM, too. HA1004, used as a negative control of H-7, failed to inhibit the growth of HuH-7 cells and the activity of hepatoma-derived growth factor. The growth of HuH-7 cells was stimulated significantly by about 40% by a protein kinase C activator, SC-9. H-7 did not suppress hepatoma-derived growth factor production in HuH-7 cells. CONCLUSIONS: These findings suggest that protein kinase C plays an important role in the growth of HuH-7 hepatoma cells and may be participated as a pathway in signal transduction of hepatoma-derived growth factor.

A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus.

Hepatoma-derived growth factor (HDGF) and HDGF-related proteins (HRP) belong to a gene family with a well-conserved amino acid sequence at the N-terminus (the hath region). A new member of the HDGF family in humans and mice was identified and cloned; we call it HRP-3. The deduced amino acid sequence from HRP-3 cDNA contained 203 amino acids without a signal peptide for secretion. HRP-3 has its 97-amino-acid sequence at the N-terminus, which is highly conserved with the hath region of the HDGF family proteins. It also has a putative bipartite nuclear localizing signal (NLS) sequence in a similar location in its self-specific region of HDGF and HRP-1. Northern blot analysis shows that HRP-3 is expressed predominantly in the testis and brain, to an intermediate extent in the heart, and to a slight extent in the ovaries, kidneys, spleen, and liver in humans. Transfection of green fluorescent protein (GFP)-tagged HRP-3 cDNA showed that HRP-3 translocated to the nucleus of 293 cells. GFP-HRP-3 transfectants significantly increased their DNA synthesis more than cells transfected with vector only. The HRP-3 gene was mapped to chromosome 15, region q25 by FISH analysis. These findings suggest that a new member of the HDGF gene family, HRP-3, may function mainly in the nucleus of the brain, testis, and heart, probably for cell proliferation.

Resistance to gap repair of the transposon Tam3 in Antirrhinum majus: a role of the end regions.

The extremely homogeneous organization of the transposon family Tam3 in Antirrhinum majus is in sharp contrast to the heterogeneity of the copies constituting many other transposon families. To address the issue of the Tam3 structural uniformity, we examined two possibilities: (1) recent invasion of Tam3 and (2) failure of gap repair, which is involved in conversion from autonomous forms to defective forms. The phylogenetic analysis of 17 Tam3 copies suggested that the invasion of Tam3 into the Antirrhinum genome occurred at least 5 mya, which is sufficiently long ago to have produced many aberrant copies by gap repair. Thus, we investigated gap repair events at the nivea(recurrens:Tam3) (niv(rec)::Tam3) allele, where Tam3 is actively excised. We show here that the gap repair of de novo somatic Tam3 excision was arrested immediately after initiation of the process. All of the identified gap repair products were short stretches, no longer than 150 bp from the ends. The Tam3 ends have hairpin structures with low free energies. We observed that the gap repair halted within the hairpin structure regions. Such small gap repair products appear to be distributed in the Antirrhinum genome, but are unlikely to be active. Our data strongly suggest that the structural homogeneity of Tam3 was caused by immunity to gap repair at the hairpins in both of the end regions. The frequency of extensive gap repair of de novo excision products in eukaryotic transposons was found to be correlated with the free energies of the secondary structures in the end regions. This fact suggests that the fates of transposon families might depend on the structures of their ends.

Structural conservation of the transposon Tam3 family in Antirrhinum majus and estimation of the number of copies able to transpose.

We have investigated the organization of the transposon Tam3 family in Antirrhinum majus. Genomic hybridization experiments and characterization of 40 independent Tam3 clones isolated from an A. majus plant revealed that the Tam3 family is quite conserved and the copy sizes are uniform. We did not find any copy with a deleted internal sequence, unlike what is usually observed in other transposons. This exceptionally conserved structure of the Tam3 family was confirmed by PCR and sequencing analyses. Sequencing analysis identified eight copies with sequences completely identical to that of the Tam3 transposase gene. These results suggested that a considerable number of autonomous Tam3 copies are present in the genome of A. majus. Among 24 copies which are surrounded by single copy regions of the genome, 14 copies are present as specific insertions in the line which we used, but absent in other lines. These copies are therefore predicted to be movable. If this ratio is the same for all Tam3 copies in a genome, then a maximum of 60% of the copies are estimated to be movable in the genome. The relatively high frequency of gene tagged by Tam3 might reflect the large number of movable copies in the genome.

Genomic organization of the 260 kb surrounding the waxy locus in a Japonica rice

The present study was carried out to characterize the molecular organization in the vicinity of the waxy locus in rice. To determine the structural organization of the region surrounding waxy, contiguous clones covering a total of 260 kb were constructed using a bacterial artificial chromosome (BAC) library from the Shimokita variety of Japonica rice. This map also contains 200 overlapping subclones, which allowed construction of a fine physical map with a total of 64 HindIII sites. During the course of constructing the map, we noticed the presence of some repeated regions which might be related to transposable elements. We divided the 260-kb region into 60 segments (average size of 5.7 kb) to use as probes to determine their genomic organization. Hybridization patterns obtained by probing with these segments were classified into four types: class 1, a single or a few bands without a smeared background; class 2, a single or a few bands with a smeared background; class 3, multiple discrete bands without a smeared background; and class 4, only a smeared background. These classes constituted 6.5%, 20.9%, 3.7%, and 68.9% of the 260-kb region, respectively. The distribution of each class revealed that repetitive sequences are a major component in this region, as expected, and that unique sequence regions were mostly no longer than 6 kb due to interruption by repetitive sequences. We discuss how the map constructed here might be a powerful tool for characterization and comparison of the genome structures and the genes around the waxy locus in the Oryza species.

Tam3 in Antirrhinum majus is exceptional transposon in resistant to alteration by abortive gap repair: identification of nested transposons.

Most transposon families consist of heterogeneous copies with varying sizes. In contrast, the Tam3 copies in Antirrhinum majus are known to have exceptionally conserved structures of uniform size. Gap repair has been reported to be involved in the structural alteration of copies from several transposon families. In this study, we have asked whether or not gap repair has affected Tam3 copies. Five Tam3 copies carrying aberrant sequences were selected from 40 independent Tam3 clones and their sequences were analyzed. Two of the five copies contain insertions in the Tam3 sequence. These two insertions, designated Tam356 and Tam661, are typical transposon-like sequences, which have terminal inverted repeats and cause target site duplication. These nested transposons were obviously associated with transpositional events, and did not originate from the gap-repair process. The remaining three copies had lost large parts of the Tam3 sequence. We could not find any relationship between the deletions of Tam3 sequence in the three copies and gap repair. PCR analysis of a Tam3 excision site in the nivea(recurrence:Tam3) mutant also showed that most of the repair events after the Tam3 excision involved end-joining. In addition to the results obtained here, among the other clones isolated, we could not find any of the internally deleted copies that comprise a major part of other transposon families. All of these data suggest that some feature of the Tam3 structure suppresses the structural alterations that are otherwise generated during the gap repair process.

Expansion of the IR in the chloroplast genomes of buckwheat species is due to incorporation of an SSC sequence that could be mediated by an inversion.

The chloroplast genomes in buckwheat species contain large inverted repeats which are at least 4 kbp longer than the majority of those in land plants. The length of the buckwheat inverted repeats was attributable to an additional region located adjacent to the borders of the small single-copy region. We have cloned and sequenced a 5. 2-kbp SmaI fragment corresponding to this extra region in the inverted repeats. A homology search revealed that the sequence of the SmaI fragment is highly homologous to one side of the small single-copy region of the inverted repeats in dicot chloroplast DNAs such as tobacco and beechdrops. Interestingly, a 3.7-kbp segment in the middle of the SmaI fragment is inserted in the opposite orientation relative to those of the other dicot species, and 17-bp direct repeats are found located at both the ends of the additional region. These results suggest that expansion of the inverted repeats in buckwheat chloroplast DNA might have been associated with an inversion.

[Morphometric study of low compliant bladder].

Low compliant bladder is an important cause of detrusor dysfunction, although its cause is unknown. Two groups of patients who developed low compliant bladder have been studied by the morphometric technique. One group consisted of patients with neurogenic low compliant bladder, and the other group consisted of patients with non-neurogenic low compliant bladder. Control materials were obtained from postmortal samples offered from the department of anatomy. Bladder wall samples were obtained during bladder augmentation surgery. Morphometric computer analysis was used to measure the proportions of connective tissue and muscle layer in the bladder wall samples. In the non-neurogenic group, there was a significant increase in connective tissue and a marked decrease in muscle layer proportion than the control group. On the other hand, there was a mild increase in connective tissue, but no decrease in muscle layer proportion was observed in patients with neurogenic low compliant bladder. Comparison of the results obtained from the two groups suggested that low bladder compliance in neurogenic patients is mainly caused by functional alteration of the bladder wall, whereas that in non-neurogenic patients is caused by an organic change of the bladder wall.

[A new method of measurement of the urinary bladder blood flow in patients with low compliant bladder].

Laser-doppler blood flowmetry, a new instrument for measurement of tissue blood flow, was used to evaluate the changes occurring in the bladder blood flow and the intravesical pressure during bladder distension in 4 patients with normal detrusor function, 4 patients with neurogenic bladder dysfunction and one patient with non-neurogenic contracted bladder. In patients with normal detrusor function and normal compliance, the bladder blood flow relatively decreased, but the intravesical pressure was not affected by the bladder distension. On the other hand, the bladder distension in patients with low compliant bladder caused a significant decrease of the bladder blood flow and marked increase of the intravesical pressure. These observations suggest that the reduction of the bladder compliance is related to the decrease of the bladder blood flow. Furthermore, the bladder over distension and the high intravesical pressure in patients with low compliant bladder are thought to induce deterioration of bladder compliance and upper urinary tract.

[Fetal genitourinary abnormalities associated with oligohydramnios].

After 16 weeks of gestation, amniotic fluid mainly consisted of fetal urine. Therefore, the association of oligohydramnios with fetal urinary tract abnormalities implies severe deterioration of renal function. The relationship of the kidney and amniotic fluid in pulmonary development has been investigated, and fetuses with oligohydramnios starting in the second trimester are considered to have uniformly fatal outcomes. We analysed underlying urological disorders, gestational age at presentation, and ultimate outcomes in 45 fetuses with severe oligohydramnios, and especially focused on clinical courses and prognosis of 7 surviving patients. Clinical and/or autopsy diagnosis included bilateral renal hypodysplasia in 20 patients, urethral atresia with/without prune belly deformity in 9, posterior urethral valve in 6, polycystic kidney disease in 4, hydrometrocolpos in 2, hereditary renal dysplasia in 2, and the others. The average gestational age at detection of severe oligohydramnios was about 30 weeks, ranging from 16 weeks in patient with urethral atresia. Urological disorders of 7 surviving patients consisted of 4 posterior urethral valves, one hydrometrocolpos, one hydronephrosis of the solitary kidney, and one bilateral megaureter. In these 7 patients severe oligohydramnios striated in the third trimester. Four patients required ventilator supports together with the administration of surfactant, but they were weaned in one to 4 days. There was no evidence of pulmonary hypoplasia on chest X-ray films. Urological emergency drainage was necessary in all patients on the day of delivery to 2 days postnatally. One patient with posterior urethral valve developed ESRF 6 months after birth. Two patients have a normal serum creatinine, but another 4 have slight elevation of SCr for their age.(ABSTRACT TRUNCATED AT 250 WORDS)

[Transurethral teflon paste injection for vesicoureteral reflux in neurogenic bladder dysfunction].

We experienced transurethral teflon paste injection for 12 refluxing ureters of 7 patients with neurogenic bladder dysfunction. Preoperative assessment of cystometry showed hypoactive bladder function with normal bladder compliance in 4 patients, and low compliance bladder (< 10 ml/cmH2O) in 1. Voiding cystography revealed grade 1 reflux in 2 ureters, grade 2 in 3, grade 3 in 2, grade 4 in 2, and grade 5 in 2. One ureter did not show reflux. Zero point two to 1.6 ml of teflon paste was injected on each ureter under cystoscopic observation. These patients were followed for a mean of 25.1 months. Reflux disappeared immediately after the first operations in all patients, however recurrence was observed in 2 ureters, in which improvement of reflux (grade 5 to 2) was achieved in 1 ureter but no improvement (grade 2 to 2) in another. Pyelonephritis was not encountered in any patients after injection. No complication was observed through the follow up period. In conclusion, we advocate that endoscopic teflon paste injection is a useful alternative to ureteroneocystostomy in the treatment of reflux in patients with neurogenic bladder dysfunction.

Light-inducible pigmentation in Portulaca callus; selection of a high betalain producing cell line.

We have established a unique betalain pigmentation system in callus cultures that originated from seedlings of Portulaca sp. 'Jewel'. Within three different 'Jewel' lines examined, one line (JR) was clearly superior with regard to callus growth rate and pigment formation. Furthermore, after ten cycles of selection of deeply colored callus patches, the selected clones contained on an average four times the amount of betalain as compared to the non-selected mother line. The colorization was induced by light, but disappeared in the dark. Pigment synthesis was detectable within 30 h after irradiation and showed positive correlation with irradiation periods.

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation.

The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA.

A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.

Organelle genome diversity in sugar beet with normal and different sources of male sterile cytoplasms.

Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet lines carrying normal and introduced sources of male sterile cytoplasms have been characterized and compared on the basis of restriction enzyme analysis. Normal cytoplasm was shown to contain mt and ctDNAs which differed from those of the male sterile cytoplasms examined in the present investigation. On the other hand, four groups of male sterile cytoplasms could be differentiated by their own characteristic mtDNA digest patterns, while two were separated by ctDNA comparisons. In addition, a greater degree of variability of the mitochondrial genome is suggested. Our results also imply strict maternal inheritance of mt and ctDNAs. Thus, the organelle DNA assay provides a positive and alternative means of identifying various male sterile cytoplasms.