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S Hiraoka, H Ando, M Ban, H Ueda, and A Urano


We analyzed changes in the hypothalamic levels of vasotocin (VT) and isotocin (IT) mRNA in chum salmon during spawning migration to the Ishikari river. The fish were caught at Atsuta, a fisherman's village facing the Ishikari bay, and at Chitose, an upstream branch of the Ishikari river. The former are referred to as sea water (SW) fish, and the latter as freshwater (FW) fish. The levels of VT and IT mRNA in the forebrains were determined by quantitative Northern blot analysis using single-stranded DNA with the same mRNA sequences as the standards. Levels of VT mRNA were higher in the FW males than the FW females, although no such difference was seen in the SW fish. Changes in the levels of VT mRNA were markedly different in males and females. In the males, no significant differences were seen in the levels of VT-I and VT-II mRNA between the SW and FW fish. However, in the females, the levels of VT mRNA in the FW fish were significantly lower than those in the SW fish. Changes in the levels of IT-I and IT-II mRNA were essentially similar in the males and females. These results suggest that the control of VT gene expression is different in males and females during spawning migration, although the neuroendocrine mechanism is not known.

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S Taniyama, T Kitahashi, H Ando, M Ban, H Ueda, and A Urano

Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration.

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T. Noce, H. Ando, T. Ueda, K. Kubokawa, T. Higashinakagawa, and S. Ishii


A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using λ gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-β subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0·8 and 1·0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 °C. In-situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-β cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-β subunit supports the hypothesis that the number of proline residues increases in the LH-β subunit the closer phylogenetically the vertebrate is to mammals.

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S Ueda, R P Heeley, K. R Lees, H L Elliott, and J M C Connell


A polymorphism of the gene encoding the human angiotensin I-converting enzyme (ACE), which is defined by an insertion/deletion polymorphism in intron 16, has been identified as a candidate genetic locus in the development of cardiovascular and renal disease.

We have demonstrated that the accuracy of ACE genotyping is critically dependent on the strategy of the PCR used in typing. Of 1238 individuals genotyped by a standard method, 335 were typed as DD, 645 as DI and 258 as II. However, when DD individuals were retyped using modified methods (including either 5% dimethyl sulphoxide, or a 'hot start') 35 of the original 335 samples (10·5%) were retyped as DI.

In approximately half of these mistyped samples, PCR amplification was assessed as inefficient by the absence of a third faint heteroduplex band in a control ID sample: when the assay was repeated without any modifications, the mistyped samples were correctly genotyped. In the remainder, mistyping persisted. In these cases, the use of a third 'nested' PCR primer specific for the I allele was required for successful genotyping, providing a more reliable strategy without the need for further modification to the PCR technique. Our results suggest that the triple primer approach is the method of choice for accurate ACE genotyping.