Soichi Tsuji

Genotype of stearoyl-CoA desaturase is associated with fatty acid composition in Japanese Black cattle

To investigate the genetic factors that affect fatty acid composition of beef, we compared the full-length bovine stearoyl-CoA desaturase (SCD) cDNA from 20 Japanese Black steers. Two types of the SCD gene with single nucleotide polymorphisms (SNPs) were observed in the ORF of SCD cDNA, in which an amino acid replacement from valine (type V) to alanine (type A) was predicted. We developed a method for genotyping these two SCD genes based on PCR-RFLP. We have classified 1003 Japanese Black carcasses into three genotypes, VV, VA, and AA, and compared fatty acid composition among them. The SCD type A gene contributed to higher MUFA percentage and lower melting point in intramuscular fat. The SCD genotype was not the only genetic factor contributing to fatty acid composition of Japanese Black steers, but the SCD genotype was considered one of the causes of genetic variation in fatty acid composition of Japanese Black steers. Transcription factors such as sterol regulatory element binding protein-1c (SREBP-1c) may account for the remaining part of the genetic variation in fatty acid composition.

Mitochondrial DNA reveal that domestic goat (Capra hircus) are genetically affected by two subspecies of bezoar (Capra aegagurus).

This article describes the complete sequences of the mitochondrial DNA displacement loop (D-loop) region and cytochrome b gene from domestic goats in Laos (Laos native) and wild goat “markhor” (C. falconeri). The wild goat “bezoar” (Capra aegagrus) has been considered to be the strongest candidate for the ancestor of the domestic goats (C. hircus); however, there is not sufficient molecular data to verify the hypothesis at present. In phylogenetic analyses, two wild goats, the markhor and the ibex (C. ibex), appeared as an outgroup, while the bezoar was located in a cluster of domestic goats. Mitochondrial haplotypes of Laos natives revealed two distinct major clusters: one was the same as the bezoar, the second, unique to Laos natives. The topology and calibrated levels of sequence divergence suggests that these clusters might represent at least two different subspecies of ancestral bezoars.

Development of breed identification markers derived from AFLP in beef cattle

In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. This paper describes the development of DNA markers to discriminate between Japanese Black and F1 (Japanese Black×Holstein) breeds. Amplified fragment length polymorphism method was employed to detect candidate markers absent in Japanese Black but present in Holstein. The 500 primer combinations yielded six selected markers that were converted into single nucleotide polymorphisms markers for high-throughput genotyping. The allele frequencies in both breeds were investigated for discrimination ability using PCR-RFLP. The probability of identifying F1 was 0.882 and probability of misjudgment was 0.0198. The markers could be useful for discriminating between Japanese Black and F1 and would contribute to the elimination of falsified breed labeling of meat.

Transcriptional profiling of muscle tissue in growing Japanese Black cattle to identify genes involved with the development of intramuscular fat

Japanese Black cattle are characterised by a unique ability to deposit intramuscular fat with lower melting temperature. In this study, 3 consecutive biopsies from Longissimus muscle tissue were taken and RNA isolated from 3 Japanese Black (Tajima strain) and 3 Holstein animals at age 11–20 months. The gene expression changes in these samples were analysed using a bovine fat/muscle cDNA microarray. A mixed-ANOVA model was fitted to the intensity signals. A total of 335 (4.8%) array elements were identified as differentially expressed genes in this breed × time comparison study. Genes preferentially expressed in Japanese Black are associated with mono-unsaturated fatty acid synthesis, fat deposition, adipogenesis development and muscle regulation, while examples of genes preferentially expressed in Holstein come from functional classes involved in connective tissue and skeletal muscle development. The gene expression differences detected between the Longissimus muscle of the 2 breeds give important clues to the molecular basis for the unique features of the Japanese Black breed, such as the onset and rate of adipose tissue development, metabolic differences, and signalling pathways involved in converting carbohydrate to lipid during lipogenesis. These findings will impact on industry management strategies designed to manipulate intramuscular adipose development at different development stages to gain maximum return for beef products.

Molecular weight heterogeneity of bovine serum transferrin

Cattle transferrin (Tf) was purified from serum of variant A and four bands were isolated. The peptide patterns of these bands when cleaved by proteases and by cyanogen bromide (BrCN) were compared, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Variant A displays two groups of molecules—large (L) and small (S)—on SDS-PAGE; the molecular weight of the L bands is 78,400±1700 and that of the S bands is 72,000±1700. However, S-band molecules could not be produced artificially by heat treatment of L bands in the presence of SDS and 2-mercaptoethanol. Since deglycosylated Tf also showed molecular weight heterogeneity, the sugar moieties of Tf other than sialic acids were not the cause of the heterogeneity. These results suggest that heterogeneity within a given variant is due to the presence of two kinds of molecule of different molecular weight. The peptide patterns of L and S bands produced by proteases and those produced by BrCN were distinctly different from each other. However, the stepwise degradation patterns of L and S bands resembled each other when treated with both chymotrypsin and BrCN. This suggests that L-band molecules differ from S-band molecules only in the presence of an additional carboxyl-terminal peptide.

Phylogenetical and ontogenetical studies on the molecular weight heterogeneity of bovine serum transferrin

Antitransferrin (Tf) rabbit serum was highly specific: it reacted with Tfs of ruminants, such as European breeds and Zebu breeds of cattle, Bali cattle, banteng, swamp and river types of water buffalo, anoa, goat, sheep, deer, antelope, camel, and giraffe, but did not react with serum of other non-ruminant species, such as pig, wild boar, hippopotamus, horse, rabbit, rat, chicken, etc. Electrophoresis of Tf and immunoglobulin G (IgG) complexes was carried out using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Within ruminants, the following species showed two Tf molecules on SDS-PAGE; European and Zebu cattle, Bali cattle, banteng, two types of water buffalo, and two species of anoa. Other ruminants, sheep, goat, deer, antelope, camel, and giraffe, etc., showed only one Tf molecule. The Tf heterogeneity in molecular weight was, thus, restricted to Bos, Bubalus, and Anoa. The molecular weight of Tf of water buffalo was slightly larger than that of cattle on the gel. The peptide pattern from cyanogen bromide cleavage of Tf of the water buffalo differed clearly from that of cattle. Fetal Tf showed only one molecule during development, but a newborn calf has two Tf molecules, (one large and one small) within 18 hr after birth. We suggest, therefore, that the small molecules formed during the last month of gestation. The peptide patterns of adult and fetal Tfs cleaved by cyanogen bromide differed with regard to the two large peptides; fetal Tf, lacking the second-largest peptide, had twice the amount of the largest peptide compared with adult Tf. From these results, we suggest that a change in peptide sequence occurs from the last month of gestation, when the largest peptide is degraded to the second largest. However, a Tf-like protein detected in the liver microsomal fraction has only one molecular size, both in adult and in fetal livers.

Autosomal genetic control of the activity of a new variant ornithine transcarbamylase in chicken kidney

The mode of inheritance of the gene for chick kidney ornithine transcarbamylase (OTC), found previously as a genetic variant, was investigated. White Leghorn B line males homozygous for the allele for the variant OTC gene were selected using the California Gray breed, having a near-absolute deficiency of the enzyme. Then further crosses of the two breeds were made. The mean value of the OTC level of F1 progeny was about 170 units. Chicks from the backcross generation were divided into two groups, of high activity and low activity, in a ratio of 1:1. F2 chicks were divided into three groups: one-fourth of the chicks were classified as a “super high” group, one-half were “high,” and the remaining one-fourth were “low” the mean values for OTC level were 356.7, 196.4, and 15.6 units, respectively. From these results, it was suggested that the variant OTC represents a simple autosomal incompletely dominant trait.

Incomplete protection mechanism against vesico-ureteral reflux and hydronephrosis in the inbred mouse strain DDD

We have previously reported the occurrence and inheritance mode of hydronephrosis in the inbred mouse strain DDD. The present investigation examined the possibility that hydronephrosis may be caused by a vesico-ureteral reflux (VUR). Bladder pressure was measured under anaesthesia in 3 strains of mice (DDD, ddY and C57BL/6). VUR was demonstrated by lower bladder pressure only in the DDD strain. Loading pressure into the renal pelvis (LPP) was significantly higher in DDD than in C57BL/6 (P<0·001). Correlations between LPP and severity of hydronephrosis were significantly positive in DDD and ddY, indicating that mice showing higher LPPs had the severest disease. Scanning electron micrography revealed that the ureteral orifice of DDD (100-300 μm) was much larger than in C57BL/6 (30 μm), and thus offered scant protection against VUR in DDD. These results suggest that DDD hydronephrosis caused by VUR is related primarily to the absence of an adequate protection mechanism in the ureteral orifice.

Chicken Ornithine Transacarbamylase: Its Unexpected Expression

Ornithine transcarbamylase (OTC), one of the enzymes of the urea cycle, is detectable in some strains of chickens, although they have no functional urea cycle. The enzyme consists of three identical subunits of 36 kd and is present in mitochondria of the kidney. Using immunoabsorbent column chromatography, we found further evidence that the enzyme is detectable as a precursor form (40 kd) in chicken brain, heart, liver, pancreas, gizzard, small intestine, and breast muscle. When an extract of small intestine containing only precursor OTC was treated with a kidney extract, the precursor was converted into OTC. This suggests that there is a tissue-specific processing protease in the kidney which splits a peptide off the precursor, causing the expression of OTC activity in this organ. However, the reason why the enzyme or its precursor is expressed in these organs is not known. The results of this study suggest that, unlike mammals, chickens are more organ specific with regard to the ability to incorporate precursor OTC into mitochondria.

Genetically controlled quantitative variation of ornithine transcarbamylase in the chick kidney

This experiment was made to show that the marked variation in ornithine transcarbamylase (OTC) activity observed within a chicken breed or among breeds is due to quantitative changes, not qualitative ones. The enzyme was partially purified from three different chicken breeds, the White Leghorn B line, the Cochin Bantam breed, and a commercial line named “G,” by the following steps: (i) extraction of OTC with Triton X-100 and cetyl-trimethylammonium bromide, (ii) heating, and (iii) salting-out column chromatography. No difference was shown immunologically, enzymologically, or physicochemically among the partially purified OTCs. The enzyme amount determined using anti-bovine OTC antiserum was related linearly to the enzyme activity either from the same chicken breed or from different breeds. These results suggest that marked variation in OTC activity reflects variation in the amount of enzyme synthesized in the kidney, and this is controlled by regulatory genes encoded on an autosome, not the structural gene.