Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome.

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.

Experience with the high occipital transcortical approach in the treatment of intraventricular hemorrhage. Report of two cases.

Two patients with intraventricular hemorrhage (IVH) were treated by direct removal of their intraventricular hematomas via a high occipital transcortical approach with successful results. This approach lies between the parietooccipital transcortical approach and the occipital transcortical approach. The patients were a 90-year-old woman with idiopathic IVH and a 60-year-old man with hemorrhage caused by bleeding in the thalamus. In both cases, the hematoma was tightly packed in the lateral ventricle. In the former case, the inferior horn of the lateral ventricle was extremely swollen, and the patient was at risk for development of uncal herniation. With the goals of complete elimination of the hematoma in the inferior horn and identification of the source of bleeding, a high occipital transcortical approach was applied, and the hematoma was removed under direct vision. With the patient in the lateral position, a minor craniotomy of approximately 3 cm was performed around the puncture site of the posterior horn (8 cm craniad from the inion and 3 cm lateral from the midline). A 1-cm cortical incision was made and the posterior horn was reached. First, the portion of hematoma at this site was removed, and then the remainder was completely removed from the interior horn and corpus. Using this method, the entire region of the lateral ventricle, including the inferior horn, corpus, and posterior horn, can be covered in a single operative field, and it is also possible to have sufficient working space for the operation.

The whorl-specific action of a petunia class B floral homeotic gene.

BACKGROUND: GREEN PETAL (GP) is thought to be a petunia class B floral homeotic gene, because the gp mutant flower displays a severe homeotic conversion of petals into sepals in the second whorl. However, since the third whorl stamens remain unaffected in the gp null mutant, gp is different from class B mutants in Arabidopsis and Antirrhinum, which also show a conversion of the third whorl stamens into the carpelloid tissue. BLIND (BL) is thought to be a petunia class A floral homeotic gene, because the bl mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures in the second whorl. RESULTS: A double mutant line homozygous for both bl and gp mutations was constructed. The bl gp double mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures with stigmatoid tips in the second whorl. In the third and fourth whorls of the mutant flower, organs remained unchanged. In the gp flower, a petunia B-type gene FBP1 is expressed strongly in the third whorl organs, but much more weakly in the second whorl organs. In the bl gp flower, FBP1 was found to be expressed strongly in the second whorl organs as well as in the third whorl organs. CONCLUSIONS: Petunia has a class B gene other than GP that determines organ identities, both in the second and third whorls of the double mutant flower, and the action of the postulated class B gene (here called PhBX) is prevented by the BL gene in the second whorl of the gp flower. PhBX appears to be a gene that specifically interacts with the FBP1 gene, and is involved in the up-regulation of FBP1.

Tnr8, a foldback transposable element from rice.

An insertion sequence 418 bp in length was found in one member of rice retroposon p-SINE1 in Oryza glaberrima. This sequence had long terminal inverted repeats (TIRs) and is flanked by direct repeats of a 9-bp sequence at the target site, indicative that the insertion sequence is a rice transposable element, which we named Tnr8. Interestingly, each TIR sequence consisted of a unique 9-bp terminal sequence and six tandem repeats of a sequence about 30 bp in length, like the foldback transposable element first identified in Drosophila. A homology search of databases and analysis by PCR revealed that a large number of Tnr8 members with sequence variations were present in the rice genome. Some of these members were not present at given loci in several rice species with the AA genome. These findings suggest that the Tnr8 family members transposed long ago, but some appear to have mobilized after rice strains with the AA genome diverged. The Tnr8 members are thought to be involved in rearrangements of the rice genome.

Gene expression of mouse S100A3, a cysteine-rich calcium-binding protein, in developing hair follicle.

We have previously identified a cysteine-rich calcium binding protein S100A3 present in the cuticle of human hair fiber. In this study, we cloned a cDNA for mouse S100A3, identified its gene location, and elucidated the expression profile throughout hair follicle development. The mouse S100A3 gene was clustered with other S100 family members on chromosome 3, and specifically expressed in dorsal skin containing hair follicles. The level of S100A3 mRNA was elevated during the anagen phase of the hair growth cycle, and sharply declined from the regression phase on. In situ hybridization revealed that the S100A3 gene was prominently expressed in cuticular cells of the hair follicle, and mRNA levels were highest in the keratogenous zone over the entire cuticular layer. Expression was also observed to a lesser extent in differentiated cortical cells; however, expression was not observed in any other component of the hair follicle or dorsal tissues. Immunohistochemical analysis showed that the S100A3 protein accumulated in cuticular and cortical cells undergoing terminal differentiation. These results indicate that the S100A3 gene is exclusively expressed, and the translation product retained, in follicular cells differentiating into major components of the hair shaft. It seems likely that S100A3 plays an important role in calcium-dependent processes leading to hair shaft formation.

Effects of tnp-470 (agm-1470) on tumor-growth, angiogenesis and serum copper levels in liver-cancer bearing rats.

Administration of TNP-470 (AGM-1470), a newly developed angiogenesis inhibitor, elevated serum copper concentrations (SCu) in a dose-dependent manner in normal rats as well as tumor-bearing rats. After discontinuation of TNP-470, SCu returned to the normal range in normal rats. In tumor-bearing rats, however, SCu decreased and then became elevated in relation to the tumor growth and capillary density in this hepatic tumor. The mechanisms leading to serum copper elevation may reflect a specific pharmacologic action of TNP-470, though it may also be related to tumor growth and reactivation of tumor angiogenesis after cessation of TNP-470.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.

Functional analysis of petunia floral homeotic MADS box gene pMADS1.

The petunia mutant green petal (gp, line PLV) shows a homeotic effect in one floral whorl, that is, the conversion of petal to sepal. We demonstrate that this mutant contains a chromosomal deletion, including the petunia MADS box gene pMADS1. Second whorl petal development in this null mutant can be restored with a CaMV 35S-pMADS1 transgene, demonstrating the essential role of pMADS1 in this process. Because gp (PLV) shows only a minor effect on stamen development, the homeotic effects of pMADS1 are different from those of B-type genes in Antirrhinum and Arabidopsis. Two other MADS box genes, pMADS2 and fbp1 (Angenent et al. 1992), require pMADS1 to maintain expression in the second whorl. However, in the absence of pMADS1 these two genes continue to be expressed in the third whorl. The functions assigned to pMADS1 are further supported by experiments in which we phenocopy gp by cosuppression of pMADS1 gene expression. The flowers, obtained through cosuppression and phenotype restoration, display different degrees of sepal to petal conversion. Analysis of these flowers indicate that pMADS1 controls growth under the zone of petal and stamen initiation, which causes the corolla tube and stamen filaments to emerge as a congenitally fused structure.

Isolation of temperature-sensitive aminoacyl-tRNA synthetase mutants from an Escherichia coli strain harboring the pemK plasmid.

The pem locus, which is responsible for the stable maintenance of the low copy number plasmid R100, contains the pemK gene, whose product has been shown to be a growth inhibitor. Here, we attempted to isolate mutants which became tolerant to transient induction of the PemK protein. We obtained 20 mutants (here called pkt for PemK tolerance), of which 9 were temperature sensitive for growth. We analyzed the nine mutants genetically and found that they could be classified into three complementation groups, pktA, pktB and pktC, which corresponded to three genes, ileS, gltX and asnS, encoding isoleucyl-, glutamyl- and asparaginyl-tRNA synthetases, respectively. Since these amino-acyl-tRNA synthetase mutants did not produce the PemK protein upon induction at the restrictive temperature, these mutants could be isolated because they behaved as if they were tolerant to the PemK protein. The procedure is therefore useful for isolating temperature-sensitive mutants of aminoacyl-tRNA synthetases.

Autoregulation by cooperative binding of the PemI and PemK proteins to the promoter region of the pem operon.

The low copy number plasmid R100 carries the pem region, consisting of two genes, pemI and pemK, which are required for stable maintenance of the plasmid. Here, to understand the regulation of the expression of the pem region, we constructed plasmids carrying either the pemI or the pemK gene, whose initiation codons were fused in frame with the lacZ gene, and examined their expression by assaying beta-galactosidase (LacZ) activity. The synthesis of both PemI and PemK proteins was found to be repressed coordinately in the presence of a plasmid carrying the entire pem region. This indicates that pemK and pemI cistrons form an operon, and that the expression of the operon is negatively regulated by its own products. We then conducted a gel retardation assay in vitro and found that the two pem products, each of which was obtained as a tripartite protein (PemI-collagen-LacZ and PemK-collagen-LacZ), bound cooperatively to a specific fragment containing the proximal region of the pem operon. The binding region, determined by DNase I footprinting analysis, included the promoter for the pem operon. This indicates that both PemI and PemK proteins bind to the promoter region to autoregulate their synthesis.

The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth.

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.

Teratogenic effect of californium-252 irradiation in rats.

The teratogenicity of californium-252 (Cf-252) irradiation which generates approximately 70% 2.3 MeV fast neutron and 30% gamma rays was evaluated. A single whole body exposure of Cf-252 at various doses was given to pregnant rats on day 8 or 9 of pregnancy, followed by microscopic autopsy of the fetuses at the terminal stage of pregnancy to search for external and internal malformations. For comparison, pregnant rats were irradiated with various doses of cobalt-60 (Co-60) standard gamma rays at the same dose rate (1 rad/min.). The doses were 20-120 rad of Cf-252 and 80-220 rad of Co-60. Using frequency of radiation induced malformations observed on day 8 of pregnancy as an index, relative biological effectiveness (RBE) of 2.3-2.7 was obtained from the straight line obtained by modifying by the least squares method the frequency curves of malformed fetuses in total implants and in surviving fetuses. The types of malformations induced by Cf-252 and Co-60 irradiation were alike. Using fetal LD50 as an index, 2.4 was obtained as RBE when irradiated on day 8 of pregnancy and 3.1 as that when irradiated on day 9. The results showed that Cf-252 had stronger a teratogenic effect than Co-60 gamma rays.

Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts.

We cloned the pem segment of plasmid R100 containing the two genes pemI and pemK, which are responsible for stable maintenance of R100 in dividing cells, into pHS1, a temperature-sensitive replication mutant of plasmid pSC101. We then examined the effect of the pem system on the maintenance of the resultant pem+ plasmid pDOM17 in various Escherichia coli host strains upon inhibition of replication of the plasmid at a high temperature. We show that the pem+ plasmid was maintained stably in the cell population and efficiently in the two hosts, km1213 (polAts) and KP64 (recA), but less efficiently in others, such as W3110, C600, P3478 (polA), and SH2743 (sfiA sfiC); the rate of cell growth was reduced at or after the time when the copy number of pDOM17 was supposed to be 0 in all of the hosts examined. We also show that a large fraction of the non-viable pDOM17-free segregant cells was produced in the former two hosts, while a smaller fraction of such cells was produced in the latter hosts, in which cell division was inhibited for several generations. Based on these results and other observations, we point out that the pemK gene product has the function not to kill the plasmid-free segregant cells, but primarily to inhibit division of these segregants. Inhibition of cell division secondarily leads to death of the plasmid-free segregants very efficiently in the two particular hosts, resulting in an apparently more stable maintenance of the pem+ plasmid in these two hosts than in others.

A case of large intestinal malignant lymphoma.

Primary malignant lymphoma of gastrointestinal tract is relatively rare and the most of it are seen in stomach or small intestine, and in Japan only 130 cases of primary large intestinal malignant lymphoma were reported from the accumulating results of the postoperative cases in the 11th Congress of the Japanese Research Society for Cancer of the Colon and Rectum. This paper describes the case report of the primary malignant lymphoma originated from the cecum, and the review of the literature. The patient was 63 year-old female, who came to this hospital for slight fever and right lower abdominal pain that was gradually increasing. After the investigation by using barium enema and the intrapelvic CT, cecum tumor was detected. The ileocecal excision was performed, and revealed the 4 X 4.5 cm tumorous type lesion of which surface was slightly irregular. Histopathologically the tumor was follicular lymphoma (partial type), medium sized cell type by the Lymphoma-leukemia Study Group (LSG) classification. After discharge, cyclophosphamide was administered by 100 mg/day for six weeks, and the sign of the recurrence has not been observed.