Cytochrome P45017~ catalyzes both 17a-hydro~~btion and 17,204de-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P45017a in swine testis, ovary, and adrenal, we undertook the simultaneous detection Of P45017a mRNA and prottin by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-bed, paraf6n-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 -) of porcine testis U5017a as DNA probe. Immunohistochemi d study was performed by employing anti-P45017a. Hybridization signals were obtained in Leydig cells of the testis, theca intern? of the Cnvian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the DNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulasa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P45017a expression at both the protein and mRNA levels in swine adrenal glands and gonads. IntroductionIn the steroid biosynthetic pathways of testes, ovaries, and adrenals, 17a-hydroxylase catalyzes the conversion of pregnenolone to 17-hydroxypregnenolone and that of progesterone to 17-hydroxyprogesterone. 17-Hydroxypregnenolone is then converted by 17,20-lyase to dehydroepiandrosterone. Although 17a-hydroxylase and 17,20-lyase activities are two distinct biochemical reactions, studies in guinea pig (1) and in swine adrenals (2) and testis (3) have shown that both activities reside in a single specific microsomal cytochrome P-450 (P-45017a). Therefore, P-45017~ lies at a very important branch point in the entire system of steroid hormone biosynthesis, i.e., bemen glucocorticoid and mineralocorticoid biosynthesis and between glucocorticoid and androgen biosynthesis. P-45017a is expressed in the testis, ovary, and adrenal gland, but it is also important to know its precise localization for a better understanding of steroid metabolism. For this purpose, techniques directed towards illustrating the enzymes specifically involved in steroidogenesis, especially immunolocalization ofsteroidogenic enzymes, are at present considered as a better method than conventional morphological examination, including electron microscopy and immunohistochemistry of steroid hormones themselves (4-10). Recently, cDNA encoding swine testis P-45017a has been characterized and oligonucleotide probes suitable for in situ hybridization were synthesized (11). In this study, we have further pursued localization...

We describe whole-body chimerism in a newborn infant with small phallus, pseudo-vaginal perineal hypospadias, and a bifid scrotum containing gonads. The human testis determining factor gene (SRY) was detected by PCR amplification. GTG-banding chromosome analysis in peripheral blood lymphocytes and cultured fibroblasts derived from right cubital skin showed a 46,XX/47,XY, +21 karyotype. Their ratios in each cell line were 294:5 and 178:7, respectively. QFQ-banding chromosome analysis documented 3 heteromorphic satellites on trisomic chromosomes 21 in the 47,XY, +21 cell line and a homozygous satellite pattern in the 46,XX cell line. Heteromorphic patterns of chromosomes 4, 13, 14, and 22 were also different between the two cell lines. To our knowledge, such disomy/trisomy chimeras have not been described previously.

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SummaryPrevious study showed that congenital isolated TSH deficiency in Japan is resulted exclusively from a G-A transition at nucleotide 145 in exone 2 of the TSH/3-subunit gene. All reported cases were from the inbred in Shikoku Island. We describe here a 10-year-old boy with hereditary TSH deficiency in the same area. The patient was born with a weight of 3,225 g to non-consanguineous parents. Evaluation at age 2 months revealed typical manifestations of cretinism without goiter. Serum T4, T3, and TSH values were 2.53 ~g/dl, 107 ng/dl, and 0.5 t~U/ml, respectively. A TRH stimulation test showed no increment of serum TSH value. Other anterior pituitary hormone levels were all within the normal range. Two oligonucleotide primers Tla and Tlb were synthesized according to the sequence data. Amplified 169 bp nucleotides in exon 2 of the TSH/3 gene with this primer set were digested with MaeI. Both the phenotypically normal brother and normal controls showed only the 169 bp fragment, whereas the proband showed 140 and 29 bp fragments and both parents showed three fragments; 169, 140, and 29 bp. These results were consistent with the point mutation of TSH/3 gene in Japanese patients with congenital isolated TSH deficiency. Our PCR method with MaeI digestion contributes to the rapid detection of the homozygous patient and the heterozygous carrier. Key Wordscongenital isolated thyroid-stimulating hormone deficiency, hypothyroidism, thyroid-stimulating hormone p chain, polymerase chain reaction (PCR) Received November 5, 1991 ; Accepted November 25, 1991. 313 314 R. MORI et al.

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the N2 or C8 positions of guanine or the N6 position of adenine. The proportion of N2 and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases—Pol η, κ, ι, and ζ and Rev1—in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA) and N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-N2-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-N2-ABA. dG-N2-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol η and κ are stronger contributors to TLS over dG-C8-ABA, and Pol κ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2′-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.