X-chromosomal localization of mammalian Y-linked genes in two XO species of the Ryukyu spiny rat.

Ryukyu spiny rats (genus Tokudaia), which are endemic to the central part of the Nansei Shoto archipelago in Japan, have unique karyotypes with odd numbers of chromosomes and no cytologically recognizable Y chromosome. The chromosome numbers of Tokudaia osimensis from Amamioshima and of Tokudaia sp. from Tokunoshima are 2n = 25 and 2n = 45, respectively, with a putative single X chromosome. The mouse X probe hybridized to the unpaired X chromosome, except for the distal part of the short arm in a female specimen of T. osimensis and in one male and one female of Tokudaia sp. Fluorescence in situ hybridization with the Tspy (testis-specific protein, Y-encoded) gene from both male and female cells of Tokudaia sp. by PCR localized Tspy to the distal part of the long arm of the X chromosome. Another Y-related gene, Zfy, from Tokudaia sp. was also localized to the same region in both species. Although the Sry gene is absent in this species, the present results suggest that the Y-chromosome segment carrying functional Y-linked genes, such as Tspy and Zfy, is translocated onto the distal part of the long arm of the X chromosome.

Characterization of Bovidae sex-determining gene SRY.

In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3' regions.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Sex determination without the Y chromosome in two Japanese rodents Tokudaia osimensis osimensis and Tokudaia osimensis spp.

Both males and females of the species of spinous country-rats (Tokudaia osimensis osimensis, T.o.o., Rodentia: Muridae), which live on Amami Oshima Island, a southern Japanese island, have 25 chromosomes. Another species of spinous country-rats (Tokudaia osimensis spp., T.o. spp., which live on Tokunoshima Island 40 km south of Amami Oshima Island, also have an odd number of chromosomes, 45. Karyotypes of males and females by the G-band method were indistinguishable in both populations. The lesser number of chromosomes (25) of T.o.o. is likely to be a result of Robertsonian fusions of 45 chromosomes of T.o. spp. that seem to be the offspring of another spinous country-rat Tokudaia osimensis muenninki (T.o.m.), which live on Okinawa Island and have 44 chromosomes including the X and Y Chrs. The lengths of the non-paired, putative X-Chr of T.o.o. and T.o. spp. occupied roughly 3.2% and 1.7% of the total lengths, respectively, hinting at translocation or exchange of a part of the X Chr and thus in violation of Ohno's Law. Southern blot analysis with murine Sry as a probe indicated that these two animals do not have Sry. When Zfx from T.o. spp. was used as a probe, both males and females of T.o.o. and T.o. spp. showed two bands, suggesting possible translocation of Zfy from the Y Chr. Comparison of physical characteristics, constituents of chromosomes, and sex-determination methods of these three Tokudaia country-rat populations suggests that each is endemic to each island and constitutes an independent species. These specialized species would provide us with clues to elucidate the mechanisms of primary sex determination and karyotype evolution in mammals.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Structural characterization of the chicken SF-1/Ad4BP gene.

SF-1/Ad4BP was identified as a master regulator controlling steroidogenic P-450 genes and belongs to the steroid hormone receptor superfamily. It is expressed in the adrenal cortex, gonads, and pituitary gonadotroph. Targeted disruption of the mouse SF-1/Ad4BP gene showed that it plays a critical role in the development of the steroidogenic tissues and pituitary gonadotroph. We have recently cloned the chicken SF-1/Ad4BP cDNA and have now cloned the chicken SF-1/Ad4BP gene and analyzed its promoter activity. This gene consists of seven exons as well as mammalian counterparts and spans about 15 kb. In mice, the gene encodes another protein, ELP, but we could not find the open reading frame of ELP in the chicken SF-1/Ad4BP gene. The promoter of this gene included five putative cis elements (E, CCAAT, GC and TATA boxes and a GA-rich element), although no TATA box has been found in mammalian counterparts. The E and CCAAT boxes moderately affected promoter activity and the GA-rich element and TATA box were essential for the expression of the chicken SF-1/Ad4BP gene.

Mea2/Golga3 gene is disrupted in a line of transgenic mice with a reciprocal translocation between Chromosomes 5 and 19 and is responsible for a defective spermatogenesis in homozygotes.

A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation. The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization. The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis, while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis. Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604 mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes.

Cloning and mapping of bovine ZFX gene to the long arm of the X-chromosome (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13), ovine (Yp12-p13), and caprine (Yp12-p13) Y chromosome.

A part of mouse Zfy-2 sequence was synthesized and used to screen a genomic library of the spinous country-rat (Tokudaia osimensis spp., 2n = 45). An isolated clone had the C-terminal region of Zfy, which consisted of 1190 bp, encoded 336 amino acid residues, and harbored 11 out of 13 zinc finger motifs. With this as a probe, a bovine testis cDNA library was screened. Two ZFX clones were isolated and their sequences combined. The short sequence, lacking part of the 5' upstream region, was amplified by PCR or RT-PCR, cloned, and sequenced. A full-length ZFX was constructed by combining these three sequences. The bovine ZFX consisted of 5328 bp and encoded 800 amino acid residues, which contained 13 zinc finger motifs. ZFX was used as a probe for fluorescence in situ hybridization and was mapped to Xq34, different from its previously reported site at Xq21-q231. A SINE (short interspersed nuclear element) sequence consisting of 188 bp was found close to the end of the 3'-untranslated region of ZFX. The SINE sequence hybridized to all bovine chromosomes. ZFY is highly homologous with ZFX and, as a result, ZFY could be mapped simultaneously. ZFY was mapped to the distal region of the short arm of the Y Chromosome (Chr) (Yp13), contradicting the previously reported position Yq1. Ovine and caprine ZFY were also mapped with bovine ZFX. Both were mapped to the distal region of the short arm of the Y Chr (Yp12-p13). Ovine ZFX was mapped to a region close to the centromere of the X Chr (Xq13).

Molecular cloning of chicken FTZ-F1-related orphan receptors.

FTZ-F1 is a member of the orphan nuclear receptors, which belongs to the steroid hormone receptor superfamily, and plays a role in the blastoderm and nervous system development in Drosophila. Recently, several FTZ-F1 family genes have been cloned in several species. SF-1/Ad4BPs have been identified as master regulators controlling steroidogenic P-450 genes in mammals and are considered to be the mammalian homologues of FTZ-F1. Moreover, SF-1/Ad4BP plays a critical role in the sexual differentiation of gonads in mammals. In vertebrates, except for mammals, the functional homologue of SF-1/Ad4BP has not been identified before. Herein, we cloned two chicken cDNAs (OR2.0 and OR2.1), which encode putative FTZ-F1 family receptors, by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). OR2.1 consists of 3255 bp, is expressed in the adrenal glands and gonads, and is considered to be the chicken counterpart of mammalian SF-1/Ad4BP. However, OR2.0 consists of 2945 bp, is expressed in the livers and the adrenal glands, and is considered to be the chicken counterpart of mouse LRH-1, which is a member of the FTZ-F1 family in mammals.

Correlation between Oscillation Modes and Penetration Rate of Protons in the Three-Phase-Liquid-Membrane Oscillation System

In the three-phase liquid-membrane system consisting of water-nitrobenzene-water, the decrease in pH of aqueous phases is found to be accompanied with the spontaneous electric oscillation of several modes. The increase rates of proton concentration in each aqueous phase are found to differ considerably in each modes of electric oscillation. Furthermore, the presence of an aqueous phase without surfactant is found to accelerate the proton penetration rate through nitrobenzene-water interface. The mechanisms for each mode of oscillation are discussed.

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (groups 1, 2A and 2B) the summary report of the 6th collaborative study by CSGMT/JEMS MMS. Collaborative Study of the Micronucleus Group Test. Mammalian Mutagenicity Study Group.

To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study. Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen). As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed. Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found. Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers. The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens. After merging, the positive rates for Groups 1, 2A and 2B were 68.6, 54.5 and 45.6%, respectively. Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals. Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds. Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones. After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 90.5, 65.2 and 60.0% for IARC Groups 1, 2A and 2B, respectively. Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity.

Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

Reversal of DNA polarity as revealed by sister chromatid exchanges in ring chromosomes.

DNA polarity at sister chromatid exchange (SCE) sites were studied in ring chromosomes from Chinese hamster cells. If the polarity of DNA strands was conserved. a double-length, symmetric dicentric ring chromosome with symmetric twin SCEs appeared after two cell cycles when a SCE occurred in S1. Indeed, approximately two-thirds of double-length. symmetric ring chromosomes belonged to this class. One-third of them, however, did not show symmetric SCEs, suggesting that polarity was not always conserved. SCE counts at centromeric regions were not high enough to account the frequency (about 1/3) of apparently inverted rejoining sites. The hypothesis that the polarity of rejoining sites is either conserved or inverted and that illegitimate rejoinings are partially repaired could explain the results. Telomere-like structures or intermediate structures during double-strand repair processes may contribute to the inverted polarity.

Achievements by CSGMT/JEMS.MMS: the Collaborative Study Group for the Micronucleus Test in the Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan.

The Collaborative Study Group for the Micronucleus Test (CSGMT) is one of the task groups in the Mammalian Mutagenesis Study Group (MMS) of the Environmental Mutagen Society of Japan (JEMS). It was established in 1982 and has made efforts to understand what the micronucleus test is, what are the advantages and disadvantages of the test as an in vivo detection system for mutagens/carcinogens, and to establish a standard protocol applicable to numerous chemicals. Members of the CSGMT have published more than 75 papers as part of collaborative studies and have contributed to the understanding of the nature of the micronucleus test and to setting guidelines for testing of medicinal and other chemicals. The CSGMT held some workshops to share up-to-date knowledge and techniques on the micronucleus test. Through workshops and collaborative studies, the CSGMT contributed to the maintaining of a high standard of knowledge and techniques among Japanese researchers of the micronucleus test. This paper reviews achievements made by the CSGMT until now.

Entire nucleotide sequence of mitochondrial DNA of MS/Ae mice.

The entire nucleotide sequence of mitochondrial DNA of MS/Ae mice was determined. It consists of 16,300 bases, with 15 sites being different from the known 16,295 base sequence of mitochondrial DNA derived from L cells of C3H mice (accession no. V00711). The MS/Ae strain is a derivative of CD-1 mice; these 15 sites in mitochondrial DNA from CD-1 mice were also determined. No difference was found, strongly suggesting that mitochondria of MS/Ae and CD-1 mice have the same DNA sequence and indicating that the high sensitivity of MS/Ae mice to mutagens compared to CD-1 mice is not dependent on genes coded by mitochondrial DNA. (The nucleotide sequence data in this article will appear in the DDBJ, EMBL, and GenBank nucleotide sequence database with the following accession number: D83491).

Genomic sequence analysis of the bovine male-enhanced antigen-1 (Mea-1) and differential localization of its transcripts and products during spermatogenesis.

The male-enhanced antigen-1 (Mea-1) gene was previously isolated from a bovine testicular cDNA library. In the present study, we cloned the full-length bovine genomic Mea-1 gene and compared this with the Mea-1 cDNA. The 1035-nucleotide bovine mRNA for Mea-1 (excluding the poly (A) tail) is encoded in three exons distributed over 3123 base pairs of the genome. Analysis of the 5' flanking sequence by primer extension mapping identified two main transcription start sites and several minor ones. The 5' region contained transcription-related sequences such as TATA/CAAT boxes, GC-rich regions, and several cis elements. When chloramphenicol acetyltransferase (CAT) activities of 5'-deleted clones were measured in CHO, TM4, and BALB/3T3 cells, a critical region for transcription was identified around -249 to -113 bp region from transcription start site. In situ hybridization and immunohistochemistry indicate that transcripts of the Mea-1 gene were localized in primary and secondary spermatocytes, and spermatids, but the protein products were detected only in spermatids. Intensive transcription of Mea-1 gene and specific localization of the gene product suggest that Mea-1 may play a important role in the late stage of spermatogenesis.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS.MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenesis Study Group.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS.MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

An optimal, generalized sampling time of 30 +/- 6 h after double dosing in the mouse peripheral blood micronucleus test.

Double dosing and single sampling seems to be the simplest and most reliable method for detecting clastogens in the mouse peripheral blood micronucleus test. Optimal sampling times after double dosing are studied here. Eleven clastogens [water soluble: colchicine, cyclophosphamide, cytosine arabinoside, 5-fluoro-2'-deoxyuridine, 5-fluorouracil (5-FU), 6-mercaptopurine, methotrexate (MTX), mitomycin C; water insoluble: N-2-acetylaminofluorene, diethyl sulfate, 7,12-dimethyl-benz[a]anthracene (DMBA)] were given i.p. twice to MS/Ae mice and peripheral blood samples were collected at 6 h intervals starting 24 h after the second treatment. Samples collected at 30 +/- 6 h were nearly optimal for all 11 chemicals. When DMBA, 5-FU and MTX were given, elevated micronucleus frequencies tended to last longer relative to those induced by direct-acting chemicals. Since samples collected 30 +/- 6 h after the second treatment with these three chemicals showed almost the same micronucleus frequencies observed in later samples, the time 30 +/- 6 h could be applied for these long-acting chemicals. Micronucleated reticulocytes persist in the peripheral blood (PB) longer than micronucleated polychromatic erythrocytes are retained in the bone marrow, which thus provides a simple, effective and generalized protocol for the mouse short-term PB micronucleus test, namely double dosing and sampling 30 +/- 6 h after the second dose. When one sample time has to be selected, 30 h after the second treatment is recommended.

2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) is a weak in vivo clastogen as revealed by the micronucleus assay.

The in vivo clastogenicity of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) was examined in the micronucleus test using peripheral blood from three mouse strains (ICR, CD-1, and MS/Ae) and bone marrow from one rat strain (Sprague-Dawley). Doses up to the maximum tolerated were tested. The chemical was given once, twice, thrice, or four times via either the i.p. or p.o. route. Under some conditions, ICR and CD-1 mice showed an increased frequency of micronucleated reticulocytes, but definite conclusions were difficult to draw because the increases were very slight. MS/Ae mice showed a markedly elevated micronucleated reticulocyte frequency after the double and triple ip treatments. Rats showed a slightly but statistically significantly increased frequency of micronucleated polychromatic erythrocytes after double i.p. treatments. These results indicate that AF-2 is a weak in vivo clastogen.

Cloning and sequence analysis of cDNA encoding the bovine testis-derived male-enhanced antigen (Mea).

A full-length cDNA encoding the bovine male enhanced antigen (Mea) has been cloned from a bovine testicular cDNA library and sequenced. The primary structure of the bovine Mea peptide deduced from this nucleotide sequence has 174 amino acid residues and is highly homologous to human (95.9%, 165/172) and mouse (92.5%, 161/174) Mea gene products. It is located on an autosome, and is expressed highly in the testes.