Tsutomu Hashiguchi

Genus specificity and extensive methylation of the W chromosome-specific repetitive DNA sequences from the domestic fowl, Gallus gallus domesticus

Two female-specific repeating DNA units of 0.6 kilobase pairs (kb) and 1.1 kb, produced by digesting the genomic DNA of the White Leghorn chicken with Xho I, were cloned by inserting them into the Xho I site of an Escherichia coli plasmid vector pACYC177. Two such recombinant plasmids, pAGD0601 and pAGD1101, containing a single 0.6-kb and 1.1-kb sequence, respectively, were used as molecular probes. In situ hybridization of the 3Hprobes to the metaphase chromosomes from the female White Leghorn embryos revealed their localization in the W chromosome. Semiquantitative Southern blot hybridization with 32P-probes in excess indicated that the 0.6-kb unit and 1.1-kb unit were repeated approximately 14,000 and 6,000 times, respectively, in the W chromosome. The two units comprised about 46% of the W chromosomal DNA. These two repeating units were found in the female genomes of every line of Gallus g. domesticus tested and in the female genomes of three jungle fowl species (G. gallus, G. sonneratii, and G. varius) but not in three species belonging to other genera in the suborder Galli. Hha I sites in the 0.6-kb and 1.1-kb repeating units were shown to be extensively methylated and a significant fraction of the Hpa II sites in the 0.6-kb repeating units were also shown to be methylated in the female genome of the White Leghorn. Methylation patterns of Hpa II sites in or around the 0.6-kb repeating units examined by the Msp I digestion were similar in the various lines of domestic fowls and the two species of jungle fowls, but G. varius (black or green jungle fowl) produced a different pattern of digestion with Msp I.

Variation among chicken stocks in the fractional rates of muscle protein synthesis and degradation.

Fractional rates (% · day−1) of synthesis and degradation were determined by measuring the output of Nτ-methylhistidine (MeHis) in the excreta at 4 and 8 weeks of age in the chicken. At 4 weeks of age, the fractional rate of synthesis of the meat-type stock was twice that of the egg-type stock (White Leghorn), but the fractional rates of synthesis at 8 weeks of age were similar (4.1–5.1% · day−1) among stocks. The fractional rate of degradation (1.3–1.5% · day−1) of the meat-type stock at 8 weeks of age was less than half the rate of the egg-type stock (2.9% · day−1). The fractional rates of synthesis and degradation at 4 weeks of age in the Satsuma native fowl were relatively high compared with those in the other stocks. In particular, the rate of degradation (8.6% · day−1) at 4 weeks of age was approximately twice that of other stocks. These results show that fractional rates of synthesis and degradation of muscle protein in the chicken differ among genetically diverse groups. The effect of changes in rates of synthesis and degradation on the change in fractional growth rate also differed. From regression coefficients (bKs· FGR and bKd· FGR) of these rates in skeletal muscle protein on the fractional growth rate, it was recognized that the change in growth rate accompanies the changes in both synthesis and degradation in White Leghorn and commercial broilers but only the change in synthesis in White Plymouth Rock (dw) and Satsuma native fowl.

Genetic studies on the muscle protein turnover rate of coturnix quail.

The validation of the urinary excretion of Nτ-methylhistidine (Nτ-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-14C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-14C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-14C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that Nτ-MH released from myofibrillar protein is not reutilized and the excretion of Nτ-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary Nτ-MH excretion and fractional synthesis (Ks) and degradation (Kd) rates at the high growing period were higher than those at the low growing period. The Ks and Kd are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.

Comparison of calpain and calpastatin activities in skeletal muscle of broiler and layer chickens

1. The objective of this study was to estimate the difference between broiler and layer chicks in the activities of calpain and calpastatin (inhibitor of calpain) in breast muscle. Differences between broilers and layers in body weight, daily gain at 3 weeks of age and fractional growth rate (FGR) during 2 and 3 weeks of age were statistically significant (P < 0.01). 2. Calpain and calpastatin activities were measured at three weeks of age with alkali-denatured casein as a substrate. The m-calpain (calpain activated by millimolar calcium concentration) activities in units/g muscle and units/mg extractable muscle protein were 0.779 and 0.353 for broilers, and 1.042 and 0.440 for layers, respectively. The calpastatin activities in units/g muscle and units/mg extractable muscle protein were 0.332 and 0.153 for broilers, and 0.262 and 0.112 for layers, respectively. 3. Broilers with high FGR showed low m-calpain and high calpastatin activities. In contrast, layers with low FGR showed high m-calpain and low calpastatin activities. 4. These results suggest that m-calpain and calpastatin activities in skeletal muscle vary between breeds which have different rates of muscle production.

Comparison of energy metabolism during the growing period in quail lines selected for body weight

1. The present study was conducted to compare body weight, daily weight gain, relative growth rate, food intake, food conversion efficiency, abdominal fat weight, thyroid weight, plasma T4 concentration, body temperature, oxygen consumption, carbon dioxide production and heat production among three Japanese-quail lines selected for body weight: a random bred line (RR) and lines for large (LL) or small (SS) body weight. 2. T4 concentration increased in the order SS, RR, LL in both sexes and did not vary significantly between sexes; the SS line had a significantly higher value than that of the LL line. 3. The body temperatures of SS, RR and LL lines were 42.45, 42.03 and 41.25 degrees C in males, and 42.7, 42.03 and 41.63 degrees C in females. 4. The oxygen consumptions of SS, RR and LL lines were 63.4, 46.0 and 43.8 ml/kgW0.75 min. in males, and 61.4, 44.8 and 37.2 in females. The value for SS was significantly higher than those of RR and LL lines in both sexes (P < 0.01). The carbon dioxide productions of SS, RR and LL lines were 31.8, 33.8 and 27.3 ml/kgW0.75 min. in males, and 31.2, 31.9 and 27.3 in females. In both sexes, that of the LL line was significantly lower than those of the SS and RR lines (P < 0.01). 6. The heat productions of SS, RR and LL lines were 1.178, 0.994 and 0.842 kJ/kgW0.75 min. in males, and 1.142, 0.879 and 0.736 in females. In both sexes, the heat production of the SS line was higher than those of the RR and LL lines (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

GENOMIC CLONING AND PARTIAL CHARACTERIZATION OF HUMAN CHYMOTRYPSINOGEN GENE

SummaryChymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescence in situ hybridization and G-banding analysis.

The effect of the dw gene on the muscle protein turnover rate in chickens

Fractional rates (%/day) of muscle protein synthesis and degradation of the genotypes Dw/Dw and dw/dw of male White Plymouth Rock chickens were determined by measuring the output of Nτ-methylhistidine (Nτ-MH) in the excreta at 2, 4, and 8 weeks of age. The fractional growth rate of dw/dw was significantly lower (P<0.05) than that of Dw/Dw at 2 weeks of age but not at 4 and 8 weeks of age. No significant differences in the degradation rate (Kd; %/day) were found at any age. A significant difference (P<0.05) between genotypes in the rate of synthesis (Ks; %/day) was found at 2 weeks of age (Dw/Dw=11.8, dw/dw=9.9) but not at 4 and 8 weeks of age. These results suggest that the dw gene has a depressing effect on the synthesis rate of muscle protein, and the difference between genotypes in the growth rate at the early stage is a reflection of this effect.

Hormone determining the Black Pupal Colour in the Silkworm, Bombyx mori L.

IN general the pupal colour of normal strains of the silkworm, though slightly varying in shade, is amber; it can be easily distinguished from that of black pupa strains. The exocuticle of the black pupa contains much more black pigment than that of the normal pupa. From the fact that the pigment dissolves in alkaline solution, but not in acid, it is supposed to be melanin. In the silkworm, two kinds of black pupae are known. One is controlled by the bp gene, which is located on the XI chromosome, and the other is controlled by the so gene, which has no linkage with the bp gene1–3.

Variation among lines selected for body size in the fractional rate of degradation of protein and acid protease activity in the muscle of quail (Coturnix coturnix japonica)

Fractional rates (%/day) of degradation of muscle protein were determined by measuring the output of Nτ-methylhistidine (Nτ-MH) in the excreta at 2 and 10 weeks of age in three lines of quail, a random-bred line and two lines selected for body size, one for increased and the other for decreased size. In all lines, fractional rates of degradation of muscle protein at 2 weeks of age were higher than those at 10 weeks of age. The fractional rate of degradation at 2 weeks of age was highest for the RR line, 9.1–9.2%/day. However, at 10 weeks of age, the rank order changed, and the RR line showed the lowest rate, 1.8–1.9%/day. The SS line (5.8–6.2%/day at 2 weeks and 5.8–5.9%/day at 10 weeks of age) was significantly higher than the LL line (4.1–4.2%/day at 2 weeks and 2.1–2.2%/day at 10 weeks of age). Acid protease activities in supernatants of homogenized muscle of the three lines of quail at 2 and 10 weeks of age were measured. In all lines, the acid protease activities in supernatant of homogenized muscle decreased from 2 to 10 weeks of age. At 2 weeks, the protease activity of the RR line was significantly higher than that of the LL and SS lines, which did not differ significantly. However, at 10 weeks of age, the SS line had higher activity in both sexes than the LL and RR lines. The results suggest that selection for body size brings about significant changes in both fractional degradation rate and acid protease activity in the muscle.

Heterosis in body weight and muscle protein turnover rate in the coturnix quail

Taux de synthese et de degradation des proteines musculaires, poids corporel, taux de croissance et conversion de la nourriture etudies chez des hybrides heterotiques de la caille du japon obtenus par croisement entre 2 lignees endogames