3.2 BIO-MEDICAL SCIENCE Cell Biology

　Radiation effects on genetic stability would depend on features of transposons. Most eukaryotic genomes contain retrotransposons, mobile genetic elements that amplify via an RNA intermediate. Like retroviruses, these elements encode gag and reverse transcriptase (RT) genes. Long terminal repeats (LTRs), however, are not commonly present, and the elements fall into two groups, LTR and non-LTR retrotransposons. Non-LTR retrotransposons have also been called LINE-like elements because long interspersed elements (LINEs) of mammals possess a typical structure of non-LTR retrotransposons. Some LINEs representing human L1 family, characterized by frequent truncation at 5' end and the resultant short 3' side sequences resemble short interspersed elements (SINEs) in that the elements have 3' tails of poly A traces or a simple A-rich repeat. In fact, early reports of an avian LINE, CR1, had predicted it to be a SINE, in spite of the absence of the poly A tail. 　We observed that the intron of fibroin gene contains a highly abundant interspersed repetitive unit (Ichimura, S. and Mita, K.: J. Mol. Evol., 35, 123, 1992). We had sequenced a Bombyx mori genomic clone containing the unit and found a SINE-like repetitive sequence. Further sequencing of the genomic clones, however, showed that the element is a major non-LTR retrotransposon of B. .mori and evolutionally classified in the same group of LINE-like elements of Drosophila and Mosquito . The element, L1Bm, has a molecular size of 5.3kb containing gag and RT sequences with an A-rich 3'-tail. Most elements are truncated at the 5'-side and the abundant incomplete 3'-side sequences are dispersed in the genome. In the genome of B. mori some LINE-like elements, R1Bm, R2Bm, DONG and TRAS1 have been discovered. These LINE-like elements, however, are not so frequent and are liable to favor specific genomic environments. BMC1 reported as an abundant dispersed LINE-like element was a partial sequence of L1Bm and therefore, in insect L1Bm2 is the first complete unit corresponding to a major LINE in B. mori. Interestingly new LINEs have been identified recently in non-mammalian organisms, chicken, lilium speciosum and trypanosoma cruzi.

1ˉA Major LINE of Bombyx mori , L1Bm

Sachiko Ichimura, Kazuei Mita and Kimihiko Sugaya

Keywords:retrotransposon, LINE, Bombyx

　In the classical complement cascade, Cls activated by Clr cleaves C2 and C4 which are known as Cls sole substrates. Recently, however, we showed that Cls degrades type ⅲ collagen and gelatin and activates zymogen of matrix metalloproteinase-9. Type ⅲ collagen is a major constituent of cartilage matrix and MMP-9 is synthesized by chondrocytes. MMP-9 is colocalized with Cls at the ossification center. Therefore, Cls is thought to have a role in cartilage degradation. 　Cartilage consists of chondrocytes and extracellular matrix which is composed of several types of collagen and various kinds of proteoglycans. For long bone development and growth, chondrocytes pass through resting, proliferating, maturing, hypertrophic and degenerating stages. This naturally occurring cell death aids in tissue remodeling. Degenerated chondrocytes disappear leaving enlarged lacunas and surrounding matrix which is eventually degraded and the site is replaced by bone marrow. Programmed cell death of chondrocytes is accompanied by an expression of an s-myc protein and tissue transglutaminase. In most tissues dead cells are usually cleaned away by phagocytotic cells. In cartilage, an avascular tissue, however, degenerating cells disappear before monocyte-like phagocytotic cell invasion. Degrading enzymes may play a major role in the cleaning mechanisms in cartilage. We have shown that Cls synthesis increases in accordance with chondrocyte differentiation both in vivo and in vitro. Cls synthesized by chondrocytes or delivered by blood stream, may participate in chondrocytes and matrix degradation. Cls has to be activated in situ to achieve this function. In order to prove this possibility by an immunohistochemical technique,we designed inactive and active Cls spacific antibodies, RK4 and RK5, respectively. On activation, hamster Cls is cleaved between 423Arg and 424Ile generating a new NH２-terminal site of the L chain. RK5 recognizes this new epitope of the L chain and RK4 recognizes an uncleaved peptide around the cleavage site. 　Cls secreted from primary hamster embryo fibroblasts was 100◇ inactive form. On the other hand, Cls secreted from malignant hamster embryo fibroblasts was 100◇ activated. RK3, an antipeptide antibody recognizing around the active center, reacted with both active and inactive Cls. RK4 reacted only with inactive Cls. By contrast, RK5 recognized the L-chain of activated Cls but not the inactive one. Cls activation was expected to be visualized in situ using these antibodies. RK4 reacted strongly with cytoplasm of hypertrophic chondrocytes. In contrast, RK5 stained Cls in cartilage matrix around the invading vessels. In addition, we found that Cls degraded decorin which is one of the proteoglycans present in cartilage. These results strongly suggest participation of Cls in cartilage remodeling.

2ˉComplement C1s, a Classical Enzyme with Novel Functions at Endochondral Ossification Center: Immunohistochemical Staining of Activated Cls with a Neoantigen Specific Antibody.

Hisako Sakiyama Koichi Nakagawa, Kazuko Kuriiwa, Kazusi Imai１,Yasunori Okada１ and Shinobu Imajoh-Ohmi２ (１Kanazawa Univ.: ２Univ.of Tokyo)

Keywords : neoantigen specific antibody,complement Cls,cartilage, hypertophic chondrocytes, programed cell death, ultrastructure

[Publications]Publications: 
1)Toyoguchi, T., Yamaguchi, K., Imajoh-Ohmi, S., Kato, N., Kageyama, H., Sakiyama, S. Nagasawa, S., Moriya, H. and Sakiyama, H.: Biochem. Biophys. Acta, 1250, 90-96, 1995. 
2)Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K.I.P. J.and Hirabayashi, Y.: Proc. Natl. Acad. Sci. USA, 93, 4638-4643, 1996.
3)Sakiyama, H., Nishida, M., Sakai, N., Nagino, K., Miyatake, S., Saito, T., Imajoh-Ohmi, S.: Intnl. J. Cancer, 66, 768-771, 1996.
4)Toyoguchi, T., Yamaguchi, K., Nakagawa, K., Fukazawa, T., and Sakiyama, H.: Cell Tissue Res. 285, 199-204, 1996.
5)Hidari, K. I. P. J., Ichikawa, S., Fujita, T., Sakiyama, H. and Hirabayashi, Y.: J. Biol. Chem., 271, 14636-14641, 1996.
6)Sakiyama, H. Nakagawa, K., Kuriiwa, k. Imai, K., Okada, Y. Tsuchida, T., Moriya, H. and Imajoh-Ohmi, S.: Cell Tiesue Res. in Press.
7)Wada, E., Sakiyama, H., Nakamura, M. and Kanegasaki, S.: Cell Biochem. Fun., in press.

Ubiquitin is a highly conserved small protein involved in many cellular processes. Ubiquitin is thought to function in the UV-induced transcriptional induction response (UV response) by participating in the metabolism of the required transcription factors AP-1 and NF-κB. 　We previously showed that the expression of the polyubiquitin gene UbC of HeLa cells, which encodes nine tandemly repeated ubiquitins with no intervening spacer, is enhanced after UV irradiation and TPA treatment. Further, we showed that the Chinese hamster polyubiquitin gene CHUB2, encoding eight tandemly repeated ubiquitins followed by a non-ubiquitin polypeptide of 50 a.a., is an evolutionary equivalent of the human UbC gene on the basis of the sequence homology in the 5' and 3' untranslated regions. However, we observed that the expression of the CHUB2 gene is not apparently inducible by UV light in V79 Chinese hamster lung fibroblasts. No induction of the ubiquitin genes by UV light or TPA has been reported in Chinese hamster ovary cells (CHO) either. 　In order to initiate investigation of the mechanisms behind the dissimilar regulation of the Chinese hamster CHUB2 gene and the human UbC gene, we determined the nucleotide sequence up to -4345 bp from the transcriptional initiation site of the CHUB2 gene (deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number D63782), and compared it with that of the human UbC gene. As shown in Fig.1, the 5' control region of the CHUB2 gene is not characterized by clustered recognition sequences for transcription factors as seen in the UbC gene. There are no AP-1 sites, few NF-κB sites and even fewer SP-1 sites, reflecting the low GC content of this region compared with the corresponding region of the human UbC gene. It is considered likely, from this result, that the absence of AP-1 sites in the less organized 5' control region of the CHUB2 gene may well be responsible in part for the dissimilar regulation of the two genes by UV light and TPA, despite the fact that these genes are evolutionarily equivalent.

3ˉComparison of the 5' Upstream Region of the Evolutionarily Equivalent Polyubiquitin Gene of Humans and Chinese Hamsters

Mitsuru Nenoi, Kazuei Mita, Sachiko Ichimura and Iain L. Cartwright■ (■Univ. of Cincinnati, U.S.A.)

Keywords:polyubiquitin gene, evolutionarily equivalent

[Publications]Nenoi, M., et al.: Gene, 179, 297-299, 1996.
Fig.1 Schematic representation of overall structure of the UbC and CHUB2 genes. Sites of a perfect match to the consensus sequences for transcription factors AP-1, NF-κB, SP-1, NF-IL6, HSF and RAD sequence in the upstream region and around the transcription initiation site of each gene are indicated, together with the TATA box. The numbers given in the hatched coding region boxes indicate the individually encoded ubiquitins. The darkly shaded box in the 3' coding region of the CHUB2 gene represents the non-ubiquitin polypeptide of 50 a.a.

　The nucleotide sequence of the polyubiquitin gene UbC of HeLa S3 cells and its upstream region were determined and characterized. Recognition sequences for the transcription factors HSF, NFκB, AP-1(c-jun), NF-IL6 and Sp1 were found in the upstream control region, a result consistent with the observation of a distinct regulatory response for the UbC gene compared with that of another polyubiquitin gene UbB. Employing a PCR procedure to amplify the entire coding region from genomic DNA, we found a heterogeneity in the repeat number (eight and nine repeats) of the ubiquitin coding units, which resulted from an apparent deletion of either the seventh or the eighth unit in the predominant nine-ubiquitin-unit coding gene. In addition, by comparison with the nucleotide sequence of the UbC gene of human leukocytes previously determined, we found a significant number of nucleotide discrepancies. However these discrepancies could be substantially reduced by realigning the units so that the first and second ubiquitin units of the sequence determined here are translocated to the boundary between the eighth and the ninth units.

4ˉHeterogeneous Structure of the Polyubiquitin Gene UbC of HeLa S3 Cells

Mitsuru Nenoi, Kazuei Mita, Sachiko Ichimura, Iain L. Cartwright１, Ei-ichi Takahashi２, Masatake Yamauchi and Hideo Tsuji (１Univ. of Cincinnati, U.S.A.; ２Otsuka Pharmaceutical Co.)

Keywords:variable number of tandem repeat, unequal crossover, polyubiquitin gene

[Publications]Nenoi, M., et al.: Gene, 175, 179-185, 1996.

　The ultimate function of the mammary gland is to produce milk which contains important substances for newborns, and it is known that the growth and differentiation of the mammary gland are basically controlled by multiple interactions of several peptide and steroid hormones from endocrine organs, such as the pituitary and ovary. Casein is one of the major components of milk which is produced and secreted by the mammary gland epithelium in a hormonally controlled manner during the lactational period. Indeed, appreciable amounts of casein mRNAs are present in the rat mammary gland during pregnancy and lactation. Subsequently, casein as well as α-lactalbumin represent specific molecular markers of the secretory activity, and the degree of differentiation of epithelial cells in the mammary glands. 　On the other hand, the determination of casein by radioimmunoassay in the serum of patients with various malignancies, not only of the breast but also of other organs, raised concern about the specificity of casein production as a marker of the mammary gland origin. In this context, we have attempted to clarify the presence of caseins and casein-like proteins in a variety of organs, since caseins could be a useful molecular marker to understand the secretory function and the differentiation mechanisms of epithelial cells not only in the mammary gland but in many other organs. 　Casein-like proteins were detected in various organs of rat by using a specific antiserum raised against rat milk caseins. The antiserum specifically recognized α１-, α２-, β- and γ-caseins in the rat milk by Western blot analysis. Immunohistochemical studies of this antiserum on the formalin fixed mammary glands showed that immunoreactive caseins localized on the apical portion of cytoplasms of lactating mammary epithelial cells and in the secretion of lumen, which indicates a directional secretion of caseins to the lumen from the mammary epithelial cells. Immunoreactive substances using this antiserum were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of the adrenal gland, distal tubes and convoluted collecting tubes of the kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands and the pickle cell layer of the skin, uterine glands and epithelium of the endometrium, hepatic bile ducts and the brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to α２-casein of rat milk. Skin was the only tissue which expressed both α２- and β-caseins. These findings suggest that the α２-casein-like substance is not only localized in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs. 　One noteworthy finding was that the casein-like substances localized in the epithelium of exocrine ducts in a variety of organs, such as pancreas, salivary gland, kidney and liver. In addition, the hormonally controlled organs including the adrenal gland and uterus also expressed the casein-like substances. Another observation in this study was that the α２-casein-like protein was a major component in the casein-like protein expressed by organs. In our understanding, this is the first time that the α２-casein molecular species was identified in various organs by Western blot analysis with a specific antiserum. One possible explanation for this phenomenon is the hormonal regulation of casein expression in a variety of organs. It is well understood that the syntheses of the caseins and their mRNAs are dependent upon hormonal control. A significant level of mRNAs for caseins is found in virgin rat mammary glands in which α-casein mRNA is present more than β- and γ-casein mRNAs. The concentration of mRNA for β-casein becomes higher than that of other casein mRNAs after gestation. Therefore, in our study, it is likely that the γ-casein-like protein was not detected by immunoblot analysis in the various organ extracts obtained from non-pregnant rats, and that most of these organs were abundant in the α-casein-like protein (especially, α２-casein-like protein) except the salivary gland. These observations may reflect the hormonal regulation of casein-like proteins in various organs as well as in the mammary gland. 　Although the localization of caseins in a variety of organs is now certain, the physiological roles of casein and casein-like proteins remains obscure. There are interesting reports which imply some possible roles of caseins. Cytotoxic T lymphocytes (CTL) expressed members of the casein gene family, such as α-, β- and κ-caseins. Casein micelles act as a vehicle by which perforin, an important cytolytic mediator released from CTL upon antigenic stimulation, is delivered onto the surface of target cells. Furthermore, low levels of mRNA for α-casein was detected in thymus by Northern blot analysis after PCR amplification. This is consistent with our finding that thymus extract contained α２-casein-like substance. Given these observations, caseins may be important as a sort of carrier protein in thymus, and CTL function. 　Although the specific anti-rat casein antiserum we generated provides a useful tool to analyze the mode of expression of the respective genes by not only immunnohistochemistry but by Western blotting, cloned cDNA probes in general would be superior to immunological methods for the detection of specific markers in various organs, therefore, the recognition of mRNAs for caseins or casein-like substances by specific cDNA probes would be better approach to clarify the presence and localization of casein-like proteins in various organs. Furthermore, certain specific cDNAs will provide better understanding with regard to the regulation mechanisms of respective genes by steroid and peptide hormones in various organs.

5ˉDistribution of Casein-like Proteins in Various Organs of Rat

Makoto Onoda and Hiroshi Inano

Keywords:casein, mammary gland, immunohistochemistry

[Publications]Onoda, M. and Inano, H.: J. Histochem. Cytochem.
 (in press)

　Twenty-five temperature-sensitive mutants were isolated from Chinese hamster CHO-K1 cells after mutanization with N-methyl-N'-nitro-N-nitrosoguanidine. One of the mutants, tsTM4, exhibited abnormal induction of sister chromatid exchanges (SCEs) along with the decrease of DNA synthesis in the cells arrested in S phase at the non-permissive temperature. The cloning of the human gene complementing the defect of tsTM4 was performed. Then, we have isolated the gene for the human RNA polymerase ⅲ largest subunit (Rpⅲ LS) and determined its genomic organization. The genomic fragments containing the whole coding regions of the human Rpⅲ LS complemented the genetic defect of tsTM4. The Northern analysis showed that both Rpⅲ LS genes (hamster and human) were transcribed at the non-permissive temperature in the transformant. These facts strongly suggest that a genetic defect in the Rpⅲ LS gene would be responsible for the abnormal induction of SCEs in the tsTM4. To search for mutations in the Rpⅲ LS gene, we have analyzed the whole region of the Rpⅲ LS cDNAs derived from tsTM4 and CHO-K1 cells, and identified the mutation site by determining the sequences completely (Fig.1).

6ˉCloning and Sequencing for the Largest Subunit of Chinese Hamster RNA Polymerase ⅲ Gene

Kimihiko Sugaya, Shun-ichi Sasanuma,Junko Nohata, Terumi Kimura, Hideo Tsuji, and Kazuei Mita

Keywords: gene structure, RpII, RT-PCR, SCE, mutation site

Fig.1. Cloning of Chinese hamster RNA polymerase ⅲ largest subunit. The black boxes (I-ⅲ) in the schematic diagram of the Rpⅲ LS gene product represent the regions conserved between the largest subunit of eukaryotes and β' subunit of E. coli RNA polymerse. The hatched box (CTD) represents the carboxy-terminal heptapeptide repeat domain. The mutation site for tsTM4 is indicated by an asterisk. Location of cDNA clones are indicated above the schematic diagram. Sequenced regions are shown as horizontal lines with vertical bars as determined terminuses. The PCR primers to clone the whole coding region of the mutant Rpⅲ LS are indicated below the schematic diagram with arrow heads for direction. 　