Evidence for cattle major histocompatibility complex (BoLA) class II DQA1 gene heterozygote advantage against clinical mastitis caused by Streptococci and Escherichia species.

Mastitis is an inflammatory response of the mammary gland to irritation, injury, or infectious agents and is a major problem in the dairy industry. We genotyped bovine major histocompatibility complex (BoLA)-DRB3 and BoLA-DQA1 genes in 120 Holstein cattle with clinical mastitis and 85 randomly selected Holstein cattle in Japan by polymerase chain reaction-sequence-based typing. The mastitis cattle were divided into four groups according to the bacterial species that caused the mastitis (Staphylococcus aureus, Streptococci, Escherichia, and coagulase-negative staphylococci). The BoLA-DRB3 and BoLA-DQA1 heterozygosity of each group was compared with that of the control cattle, while the expected heterozygosities based on Hardy-Weinberg proportions and the observed heterozygosities for each locus were compared for each group. The Escherichia-induced and Streptococci-induced mastitis groups showed significant differences between their expected and observed heterozygosities with regard to their BoLA-DQA1 genes. No differences were observed for any group with regard to the BoLA-DRB3 genes. We then found that two BoLA-DQA1 alleles promoted susceptibility to Streptococci-induced mastitis, namely BoLA-DQA1*0101 and BoLA-DQA1*10012 and that the homozygous BoLA-DQA1*0101/0101 and BoLA-DQA1*10011/10011 genotypes promoted susceptibility to mastitis caused by Streptococci and Escherichia, respectively. This is the first report showing that heterozygosity of the BoLA-DQA1 gene is associated with resistance to mastitis progression.

Microsatellite variation in Japanese and Asian horses and their phylogenetic relationship using a European horse outgroup.

The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average heterozygosities (0.75-0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing our assumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating the genetic relationships between Japanese horses and Asian horses.

Sex determination by simultaneous amplification of equine SRY and amelogenin genes.

A quick method for sex determination of horses was developed. Simultaneous amplification of the equine sex-determining region of the Y chromosome gene (SRY) and amelogenin gene (AMEL) accomplished the determination of the presence of both the Y chromosome and SRY gene. In agarose gel electrophoresis, a normal stallion showed 1 SRY band and 3 AMEL (AMELX, AMELY, and AMELX/AMELY heteroduplex) bands, and a normal mare showed a single AMELX band. In XY-mares, 3 AMEL bands were detected as in a normal stallion, but no SRY band. The present method enables a quick diagnosis for XY-mare prior to cytogenetic analysis.

The cDNA sequences of equine antioxidative enzyme genes Cu/Zn-SOD and Mn-SOD, and these expressions in equine tissues.

The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization.

Linear SRY transcript in equine testis.

Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice.

Chronological changes in superoxide-scavenging ability and lipid peroxide concentration of equine serum due to stress from exercise and transport.

It has been suggested that a variety of stresses on animals may accelerate their production of superoxide. Racehorses are considered to be exposed to substantial oxide stress due to transport and exercise for training and racing. To determine the effect of exercise and transport on racehorses in terms of superoxide and antioxidative ability, changes in the superoxide-scavenging ability of equine serum were observed using electron spin resonance (ESR). Changes in the concentration of lipid peroxide, which is produced in equine serum by superoxide, were also examined. The analysis revealed that lipid peroxide concentrations increased as a result of stress from exercise and transport. On the other hand, the superoxide-scavenging ability of equine serum showed a decline during transport, which is in sharp contrast to the increase seen immediately after a race due to the severe load exerted in exercise.

Cloning and characterization of the equine F18 gene, which has a novel exon.

A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA sequence contained sequence homologous to exon 3 of human F18 gene and a new exon that is not found in the human F18 gene. The equine F18 gene contains a CAG repetitive sequence (CRS) that is translated into a polyglutamine tract. The CRS was analyzed for polymorphism by PCR: four and two different alleles were observed with unrelated east-Asian and thoroughbred horses, respectively. The equine F18 gene was mapped to Xq29.1 by fluorescence in situ hybridization. The human F18 gene is also X-linked. These data strongly supported the conclusion that the clone contains the equine homologue of the human F18 gene.

Evaluation of five polymorphic microsatellite markers for typing DNA from decomposed human tissues--correlation between the size of the alleles and that of the template DNA.

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.

PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA.

The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes (AciI, BamHI, RsaI). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examined were classified into four types. Type 2 was most frequent in all breeds.

[Application of enzyme-linked immunosorbent assay (ELISA) to the medico-legal identification].

Medico-legal identification of saliva stains and bloodstains was performed by an enzyme-linked immunosorbent assay (ELISA) using a horseradish peroxidase conjugate in combination with the use of monoclonal antibodies. Activity of alpha-amylase in the stains was measured for an identification of saliva using an anti-human amylase antibody, and secretory IgA was detected for a species identification using an anti-IgA antibody. ABO and Lewis blood group antigens were detected using anti-A, anti-B, anti-H, anti-Lea and anti-Leb antibodies. After the solubilization of ABH antigens in blood stains with octyl-beta-D-glucose solution, ABO typing has been performed using fully automated system composed of a unit for blood group reaction based on ELISA and a unit for reading of result based on photometric analysis.

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse.

The noncoding region between tRNAPro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2-4 x 10(-8) per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.

Metabolism of finasteride in rat hepatic microsomes: age and sex differences and effects of P450 inducers.

1. The age- and sex-related metabolism of finasteride and the effects of P450 inducers and inhibitors were investigated using rat hepatic microsomes. 2. No marked age difference (3-13 weeks) in the rates of finasteride disappearance and the formation of 1 (omega-hydroxyfinasteride) and 4 (6 alpha-OH finasteride) was observed in the male rat. Whereas the rate of 1 formation remained about the same in male rat aged 1 year as compared with rat aged 7 weeks, a 21 and 45% decrease in the rate of finasteride disappearance and 4 formation, respectively, were observed. 3. The rates of finasteride disappearance and metabolite formation 1 and 4 in the female rat decreased with an increase in age (3-7 weeks). Metabolite 4 was hardly formed by the hepatic microsomes from the female rat at 7 weeks of age. 4. Hepatic microsomes from the male rat treated with phenobarbital (PB) and dexamethasone (Dex) increased the rate of the finasteride disappearance (PB, 5.5-fold; Dex, 11.6-fold), whereas no increase in this activity was observed after administration of beta-naphthoflavone (BNF). Similarly, pretreatment of the female rat with PB and Dex resulted in increases of 26.6 and 8.4-fold in the rate of finasteride disappearance, respectively, whereas no inductive effect on this activity was observed in the BNF-treated female rat. 5. These observations suggest that finasteride is metabolized by P4502B, P4502C, and P4503A subfamilies in the male rat and by P4502B and P4502C subfamilies in the female rat.

Polymorphic sequence in the D-loop region of equine mitochondrial DNA.

The D-loop regions in equine mitochondrial DNA were cloned from three thoroughbred horses by polymerase chain reaction (PCR). The total number of bases in the D-loop region were 1114 bp, 1115 bp and 1146 bp. The equine D-loop region is A/T rich like many other mammalian D-loops. The large central conserved sequence block and small conserved sequence blocks 1, 2 and 3, that are common to other mammals, were observed. Between conserved sequence blocks 1 and 2 there were tandem repeats of an 8 bp equine-specific sequence TGTGCACC, and the number of tandem repeats differed among individual horses. The base composition in the unit of these repeats is G/C rich as are the short repeats in the D-loops of rabbit and pig. Comparing DNA sequences between horse and other mammals, the difference in the D-loop region length is mostly due to the difference in the number of DNA sequences at both extremities. The similarities of the DNA sequences are in the middle part of the D-loop. In comparison of the sequences among three thoroughbred horses, it was determined that the region between tRNA(Pro) and the large central conserved sequence block was the richest in variation. PCR primers in the D-loop region were designed and the expected maternal inheritance was confirmed by PCR-RFLP (restriction fragment length polymorphism).

A novel tRNA species as an origin of short interspersed repetitive elements (SINEs). Equine SINEs may have originated from tRNA(Ser).

Short interspersed repetitive elements (SINEs) were isolated from the equine genome and characterized. The equine SINE (ERE-1) family has several features characteristic of tRNA-derived retroposons. The five members of the equine family of SINEs are approximately 230 nucleotides in length and terminate with a sequence rich in oligo(A). They are all flanked by direct repeats at the 5' and 3' ends, and such repeats are the hallmarks of retroposons. In addition, the ERE-1 family has a tRNA-related region, which is similar to tRNA(Ser) of Drosophila (65% identity). tRNA(Ser) is a novel tRNA with respect to the origin of SINEs and has not previously been recognized among the twenty tRNA-derived SINEs characterized to date. The members of the ERE-1 family were found to be distributed among five species in the genus Equus, and their amplification may have contributed to the genetic variability of their hosts during evolution.