The SNP c.1326T>G in the non-SMC condensin I complex, subunit G (NCAPG) gene encoding a p.Ile442Met variant is associated with an increase in body frame size at puberty in cattle.

Recently, we had located a bovine carcass weight QTL, CW-2, to a 591-kb interval on BTA6 and have identified the SNP c.1326T>G in the NCAPG (non-SMC condensin I complex, subunit G) gene that leads to the amino acid change p.Ile442Met in the NCAPG protein, which is a candidate causative variation. Here, we examined the association of the NCAPG:c.1326T>G locus with linear skeletal measurements of growth-associated traits during adolescence, which is a period of intensive growth, using two historically and geographically distant cattle populations: 792 Japanese Black steers and 161 F(2) bulls of an experimental cross from Charolais and German Holstein. In both populations, the SNP NCAPG:c.1326T>G was associated with each component of body frame size: height, length and width at puberty. The associations of CW-2 with height- and length-associated traits were observed at an earlier growth period compared to the associations with thickness- and width-associated traits, indicating that the primary effect of the CW-2 QTL may possibly be exerted on skeletal growth. The significant associations of the NCAPG:c.1326T>G locus with growth-associated skeletal measurements are similar to the effects of the syntenic region on human chromosome 4 that are associated with adult height in humans, supporting the hypothesis that CW-2 is analogous to the human locus and pointing to a conserved growth-associated locus or chromosomal region present in both species.

Differentially expressed genes during bovine intramuscular adipocyte differentiation profiled by serial analysis of gene expression.

Beef marbling or intramuscular fat deposition is an economically important carcass trait in Japanese Black cattle. To investigate genes involved in intramuscular adipogenesis, differential gene expression during adipogenesis in a clonal bovine intramuscular preadipocyte (BIP) cell line was profiled with serial analysis of gene expression (SAGE). We sequenced 75 283 tags for the proliferation phase (day 0) and 81 878 tags for the differentiation phase (4 days after adipogenic stimulation: day 4). A comparison of the unique SAGE tag frequencies between the day 0- and day 4-libraries revealed that 878 (2.8%) of the 30 989 unique putative transcripts were expressed at significantly different levels (P < 0.05); 401 tags (1.4%) were up-regulated and 477 tags (1.2%) were down-regulated in the day 4-library relative to the day 0-library. We confirmed up-regulation of 10 tags of the genes that were up-regulated in the previous subtraction cloning studies in BIP cells [Animal Science Journal, 76 (2005) 479]. Of the 878 differentially expressed tags, 377 were identified in the bovine RefSeq library and 356 were assigned a bovine draft genomic sequence. Fifteen tags were mapped in previously detected beef marbling quantitative trait loci (QTL) regions [Mammalian Genome, 18 (2007) 125]. These genes may be involved in the adipogenic processes of beef marbling.

Identification of a 3.7-Mb region for a marbling QTL on bovine chromosome 4 by identical-by-descent and association analysis.

QTL mapping for growth and carcass traits was performed using a paternal half-sib family composed of 325 Japanese Black cattle offspring. Nine QTL were detected at the 1% chromosome-wise significance level at a false discovery rate of less than 0.1. These included two QTL for marbling on BTA 4 and 18, two QTL for carcass weight on BTA 14 and 24, two QTL for longissimus muscle area on BTA 1 and 4, two QTL for subcutaneous fat thickness on BTA 1 and 15 and one QTL for rib thickness on BTA 6. Although the marbling QTL on BTA 4 has been replicated with significant linkages in two Japanese Black cattle sires, the three Q (more marbling) haplotypes, each inherited maternally, were apparently different. To compare the three Q haplotypes in more detail, high-density microsatellite markers for the overlapping regions were developed within the 95% CIs (65 markers in 44-78 cM). A detailed haplotype comparison indicated that a small region (<3.7 Mb) around 46 cM was shared between the Qs of the two sires, whose dams were related. An association of this region with marbling was shown by a regression analysis using the local population, in which the two sires were produced and this was confirmed by an association study using a population collected throughout Japan. These results strongly suggest that the marbling QTL on BTA 4 is located in the 3.7-Mb region at around 46 cM.

Linkage disequilibrium structures in cattle and their application to breed identification testing.

We examined the extent of linkage disequilibrium (LD) block lengths in four breed populations: Japanese Black, Angus, Hereford and Holstein. Three chromosomal regions in which QTL were previously mapped in Japanese Black populations were scanned with 84 microsatellite markers. The estimated LD lengths in these four purebred populations varied from 535 to 683 kb, which is much shorter than the values reported previously. Our findings suggest that QTL can be mapped in sub-centimorgan regions in these populations using an LD-mapping method. We also developed breed identification methods to distinguish Japanese Black from Angus, Hereford, Holstein and F(1) animals (Japanese Black x Holstein) respectively using the haplotypic frequencies of a pair of markers in the breed populations. After assessing the distributions of posterior probabilities to be Japanese Black, we obtained several pairs of markers that completely distinguished Japanese Black from the other breeds. We also obtained several combinations of six markers that completely distinguished Japanese Black animals from F(1) animals.

Mapping, expression, and association study of the bovine PSMC1 gene.

The 26S ATP-dependent protease is composed of a 20S catalytic proteasome and two PA700 regulatory modules; it plays a central role in many regulatory pathways, such as cell cycle regulation, differentiation, and apoptosis. The PA700 complex is composed of multiple subunits, including at least six related ATPases and approximately 15 non-ATPase polypeptides. PSMC1 (proteasome 26S subunit, ATPase, 1) is one of these ATPases. In this study, we amplified a fragment of 507 bp from intron 9 of the bovine PSMC1 gene and found a SNP (G/A) at position 216 in the PCR fragment. Genotyping of 138 animals from four beef breeds revealed that the average frequency for allele A (G-base) was 0.4271 (0.3269-0.5517); for allele B (A-base) it was 0.5729 (0.4483-0.6731). This SNP is significantly associated with average daily feed intake (P < 0.01), average daily gain, finishing average daily gain, body length, ratio of feed to meat, backfat thickness, and loin-muscle area (P < 0.05). Our experimental data showed that animals with an AA genotype have a significantly lower food intake, grow faster, are longer in the body, and have less backfat and bigger loin muscle; hence, their ratio of feed to meat is significantly lower. We believe that the PSMC1 SNP is a potential candidate marker for marker-assisted selection in these traits. We also found that the bovine PSMC1 gene was expressed mainly in lung, testis, and spleen. In addition, we mapped the bovine PSMC1 gene on BTA10 by an RH mapping method.

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20-specific DNA library.

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.

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.

Mapping of 1400 expressed sequence tags in the bovine genome using a somatic cell hybrid panel.

A bovine/hamster hybrid cell panel consisting of 30 independent hybrids was developed to locate genes. Polymerase chain reaction analysis of 279 microsatellites on the cattle linkage map in this panel revealed the presence of all chromosomes in either entire or fragmented form. Among primer pairs prepared from bovine 3'-expressed sequence tags (ESTs), 1400 ESTs were assigned to specific chromosomes, of which 1303 were newly assigned in this study, and mapped 854 (61%) to 1 of 192 chromosomal segments using this panel. The regional mapping of new genes to cattle chromosomes can be rapidly achieved using this panel.

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs.

We determined 36,310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16,358 non-redundant ESTs were derived from approximately 6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

Null mutation of PCLN-1/Claudin-16 results in bovine chronic interstitial nephritis.

Inherited chronic renal diseases are associated with failures in glomerular filtration and tubular resorption. Such failures invariably result from defects in selective filtration and absorption in surface renal epithelium. Recently, we described an autosomal recessive chronic interstitial nephritis with diffuse zonal fibrosis (CINF) in cattle. Bovine CINF, characterized by increased blood urea nitrogen, creatinine, and urinary proteins, leads to lethality before puberty, usually within the first 6 months or year of life. Here, we demonstrate that the first four exons of PCLN-1/Claudin-16 (CL-16), which encodes a member of the claudin family of tight junction proteins, were deleted in CINF-affected cattle. CL-16 was expressed preferentially in kidneys of normal cattle, but transcripts were totally absent in affected offspring. This observation suggests that the lack of CL-16 protein contributes to the dysfunction of paracellular renal transport systems.

Rice class III chitinase homologues isolated by random cloning of rice cDNAs.

We obtained seven kinds of cDNA clones putatively identified as encoding class III chitinases from cDNA libraries constructed from dichlorophenoxyacetic acid (2,4-D)- and benzyl adenine (BA)- treated rice callus. Putative amino acid sequences encoded in these cDNA clones were compared with those of known chitinases of other plants. Two clones coded for homologues that show high similarity to class III chitinases. These clones contained the common glutamic acid at the active site and were classified as true homologues. The other five clones, however, showed relatively low similarity to class III chitinases and their active sites contained aspartic acid instead of glutamic acid. These clones may correspond to relatives of a super family of class III chitinases. The location of the genes coding for these homologues on the rice genome has been determined by genetic linkage analysis with restriction fragment length polymorphism.

beta-Carotene accumulation in mouse tissues and a protective role against lipid peroxidation.

Mice were supplemented with beta-carotene in beverage for 10 days. After the supplement, beta-carotene accumulated mainly in the liver and to some extent in the blood plasma, kidney and lung. The beta-carotene administration was associated with an increase in the amount of retinyl ester and retinol in the liver, but not in the amount of retinol in blood plasma. Lipid peroxidation in vivo was induced by the injection of carbon tetrachloride under the dorsal skin of mice. As an index of lipid peroxidation, thiobarbituric acid-reacting substances (TBARS) were assayed in urine and tissue homogenates. Urine and kidney TBARS were reduced by the supplementation of beta-carotene. The amounts of TBARS in kidney, liver and lung, decreased with increasing amounts of beta-carotene accumulated in these tissues, i.e. inverse correlations were obtained. These results indicate that beta-carotene can suppress lipid peroxidation in mouse tissue.

Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.

Nucleotide sequence of the rodA gene, responsible for the rod shape of Escherichia coli: rodA and the pbpA gene, encoding penicillin-binding protein 2, constitute the rodA operon.

The rodA gene, which is responsible for the rod shape of Escherichia coli, was located 5 nucleotides downstream of another rod-shape-determining gene, pbpA, encoding penicillin-binding protein 2. The coding region for the RodA protein was 1,110 base pairs in length. Two plasmids, carrying a rodA-lacZ gene fusion with and without the pbpA promoter upstream of the gene fusion, were constructed. On the basis of the difference between the expression levels of the beta-galactosidase activity dependent on and independent of the pbpA promoter, we concluded that the pbpA and rodA genes constitute a single transcriptional unit called the rodA operon.

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli.

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.