Genetic correlations between yield traits or days open measured in cows and semen production traits measured in bulls.

We used a bivariate animal model to investigate the genetic correlations between yield traits or days open (DO) as characters measured in cows and semen production traits as characters measured in bulls. Lactation records of 305-day milk, fat, and protein yields, and DO, from 386 809 first-lactation Holstein cows in Hokkaido, Japan, that calved between 2008 and 2014 were used. Semen production records were collected between 2005 and 2014 and included volume per ejaculate (VOL), sperm concentration (CON), number of sperm per ejaculate (NUM), progressive motility index of sperm (MOT), and MOT after freeze-thawing (A-MOT). Number of sperm per ejaculate was log-transformed into a NUM score (NUMS). A total of 30 373 semen production records from 1196 bulls were obtained. The pedigree file used for analysing the records was involving 885 345 animals. Heritability was estimated for VOL (0.42), CON (0.12), NUMS (0.37), MOT (0.08), and A-MOT (0.11). Weak and negative genetic correlations were recorded between yield traits measured in cows and VOL, CON or NUMS measured in bulls. Moderate and negative genetic correlations were obtained between DO and MOT (-0.42) or A-MOT (-0.43). Selection focused on MOT or A-MOT measured in bulls may therefore improve DO measured in cows.

Relationships between conception rate in Holstein heifers and cows and milk yield at various stages of lactation.

We investigated the relationships between conception rates (CRs) at first service in Japanese Holstein heifers (i.e. animals that had not yet had their first calf) and cows and their test-day (TD) milk yields. Data included records of artificial insemination (AI) for heifers and cows that had calved for the first time between 2000 and 2008 and their TD milk yields at 6 through 305 days in milk (DIM) from first through third lactations. CR was defined as a binary trait for which first AI was a failure or success. A threshold-linear animal model was applied to estimate genetic correlations between CRs of heifers or cows and TD milk yield at various lactation stages. Two-trait genetic analyses were performed for every combination of CR and TD milk yield by using the Bayesian method with Gibbs sampling. The posterior means of the heritabilities of CR were 0.031 for heifers, 0.034 for first-lactation cows and 0.028 for second-lactation cows. Heritabilities for TD milk yield increased from 0.324 to 0.433 with increasing DIM but decreased slightly after 210 DIM during first lactation. These heritabilities from the second and third lactations were higher during late stages of lactation than during early stages. Posterior means of the genetic correlations between heifer CR and all TD yields were positive (range, 0.082 to 0.287), but those between CR of cows and milk yields during first or second lactation were negative (range, -0.121 to -0.250). Therefore, during every stage of lactation, selection in the direction of increasing milk yield may reduce CR in cows. The genetic relationships between CR and lactation curve shape were quite weak, because the genetic correlations between CR and TD milk yield were constant during the lactation period.

Nongenetic effects and genetic parameters for length of productive life of Holstein cows in Hokkaido, Japan.

Records of Holstein dairy cows in Hokkaido, Japan, were used to study the effects of environmental factors on length of productive life and to estimate genetic parameters for length of productive life. Each record was assigned to 1 of 3 data sets depending on the percentage of type-scored cows in the herd. This percentage was considered to partly reflect the management policy in each herd, in particular regarding culling. The A, B, and C data sets consisted of herds with none, less than 60%, and more than 60% of type-scored cows, respectively, and included 158,719, 787,598, and 131,499 records, respectively. Analyses of length of productive life were separately carried out on each data set using the Survival Kit software (Version 5.0). Nonparametric hazards estimates and the shape parameters of the baseline Weibull distribution differed between the 3 data sets. A cow having a sire originating from the United States or Canada had a relatively lower risk of being culled than a cow having Japanese sire in data set C. However, in data set A, a cow having a Canadian sire had a higher relative risk than a cow having a Japanese sire. The herd-year variance for data set A was about twice as large as for data set C. In contrast, the sire variance for data set A was about 40% of the one for data set C. As a result, heritability varied across data sets from 0.046 to 0.134. The results of this study suggest that it is important to consider factors related to herd management policy, such as the percentage of type-scored cows, in genetic analyses on length of productive life of Holstein cows in Hokkaido, Japan.

Development of a survival model with piecewise Weibull baselines for the analysis of length of productive life of Holstein cows in Japan.

A total of 100,000 records of Holstein dairy cows in Hokkaido, Japan, were used to study the current characteristics of their length of productive life, to build up a suitable model for genetic survival analysis, and to apply the piecewise Weibull baseline model. The data for this analysis only included records of cows belonging to herds in which more than 60% of cows had type score. Compared with other studies, the proportions of cows with only 1 or 2 calvings were low (24 and 46%, respectively), indicating a very low culling rate in first or second parity in Hokkaido. The median length of productive life was about 1,250 d (3 yr and 5 mo). Four different definitions of stages of lactation were studied. The best fit of the data was obtained when stages of lactation were chosen with cutpoints at 0, 60, 250, and 350 d after calving. Whatever the definition of stages of lactation, the piecewise Weibull baseline model was better than a unique baseline model. The estimated shape Weibull parameters rho for the different parity x stage of lactation combinations varied greatly (1.0 to 5.0). The estimated hazard functions based on the piecewise Weibull baseline model were smooth and reflected the important changes in observed hazard within lactation. The results of this study are expected to contribute to the development of a genetic model for sire evaluation and to genetic improvement of length of productive life of Holstein cattle in Hokkaido, Japan.

Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1.

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.

Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor.

Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced. The P. aeruginosa frr was mapped at 30 to 32 min of the P. aeruginosa chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of E. coli RRF. In an assay including E. coli polysome and elongation factor G, purified recombinant RRF of P. aeruginosa released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr. The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E. coli and Bacillus subtilis. Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.

The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages.

phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the phi CTX phage genome. The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends. Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P. aeruginosa chromosome. In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding. These findings indicate that phi CTX is a P2-like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.

Chromosome of the enterohemorrhagic Escherichia coli O157:H7; comparative analysis with K-12 MG1655 revealed the acquisition of a large amount of foreign DNAs.

A complete Xba I and Bln I cleavage map was constructed for the chromosome of an enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain isolated from an outbreak in Sakai City, Japan, in 1996. A comparative chromosome analysis with E. coli K-12 strain MG1655 was made. The EHEC chromosome was approximately 5600 kb in length, 1 Mb larger than that of MG1655. Despite the marked difference in chromosome length, the location and direction of seven rRNA operons of the EHEC strain were similar to those for MG1655. Overall organization of genes common in both strains is also highly conserved. Chromosome expansion was observed throughout the EHEC chromosome, albeit in an uneven manner. A large portion of the chromosome enlargement was observed in the region surrounding the replication terminus, particularly in a segment containing the terA locus. Sample sequencing of 3627 random shotgun clones suggested the presence of approximately 1550 kb strain-specific DNAs on the EHEC chromosome, most of which are likely to be of foreign origin.

Functional domains of Rts1 and P1 RepA proteins for initiation of replication.

Rts1 RepA and P1 RepA are trans-acting proteins essential for initiation of replication of Rts1 and P1 plasmids, respectively. We recently found that P1 RepA bound in vitro to the Rts1 replication origin as strongly as Rts1 RepA and activated the origin in vivo. However, the ori activation was quite inefficient. This study shows that by replacing a small region of P1 RepA with the corresponding region of Rts1 RepA, the efficiency of Rts1 ori activation increased markedly. Interestingly, the same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin. Thus, a region essential for efficient activation of the replication origin was assigned to the P1 RepA molecule as well as to the Rts1 RepA molecule. The region was distinct from a domain necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the respective origin.

Purification and characterization of procytotoxin of Pseudomonas aeruginosa. Dimer to monomer conversion of protoxin by proteolytic activation.

Cytotoxin of Pseudomonas aeruginosa is a cytolytic toxin that forms a pore on the target membrane by oligomerizing into a pentamer. This toxin is produced as an inactive precursor (proCTX) and is converted to an active form by proteolytic cleavage at the C terminus. We purified proCTX to apparent homogeneity and characterized it in a comparison with the active toxin. ProCTX bound to the erythrocyte membrane but did not form an oligomer on the membrane, hence the lack of hemolytic activity in proCTX. Circular dichroic experiments showed that active and proCTX have similar beta-sheet dominant structures. Intrinsic fluorescence analysis indicated that a molecule-buried tryptophan residue(s) of proCTX was exposed to the surface of the molecule as a result of conversion to the active form. In analytical gel filtration, chemical cross-linking, and analytical ultracentrifugation experiments, dimer to monomer conversion occurred with proteolytic activation.

Interaction of P1 RepA with replication origin of plasmid Rts1: capability of an initiator protein inducing replication from a foreign origin.

Rts1 RepA and P1 RepA are trans-acting proteins essential for the initiation of replication of plasmid Rts1 and prophage P1, respectively. In this study, we found that, in vitro, P1 RepA bound to the Rts1 ori fragment and Rts1 incI fragment as strongly as Rts1 RepA. In addition P1 RepA, in trans, activated the Rts1 replication origin that was cloned in pBR322, thus allowing the ori plasmid to be maintained in a polA E. coli host. Under these conditions, however, the ori plasmid was unstable as compared with that when activated by Rts1 RepA. In addition, we found that Rts1 RepA showed no interaction with the P1 replication origin.

Gene product identification and promoter analysis of hig locus of plasmid Rts1.

The hig (host inhibition of growth) genes of plasmid Rts1 belong to the plasmid-encoded proteic killer gene family. Compared with other proteic killer genes described so far, hig is unique in that the toxic part (higB) exists upstream of the antidote gene (higA). Here we describe results of the promoter analysis of hig genes together with identification of the proteic gene products of higA and higB. Two promoters were identified in the hig locus; a stronger one, named Phig, is located upstream of higB and a weaker one, PhigA, is upstream of higA within the higB coding region. The Phig activity was negatively regulated by HigA and this regulation was augmented by HigB, whereas PhigA was not subjected to such a regulation.

A new plasmid-encoded proteic killer gene system: cloning, sequencing, and analyzing hig locus of plasmid Rts1.

A new proteic killer gene system, hig, was identified on the plasmid Rts1. The hig locus consisting of a higA and higB is directly related to the temperature sensitive host cell growth conferred by Rts1. We proved that higB encoding presumably a 92-amino-acid polypeptide inhibited segregation of plasmid free cells, and higA encoding a 104-amino-acid polypeptide suppressed the higB function both in cis and in trans.

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.

Mechanism of the cytolytic action of Pseudomonas aeruginosa cytotoxin: oligomerization of the cytotoxin on target membranes.

Pseudomonas aeruginosa cytotoxin (CTX) is thought to be a pore-forming polypeptide of 29 kDa. To study whether CTX assembles into oligomer on target membranes, we solubilized membrane-bound toxin with 1% sodium dodecyl sulfate (SDS) at 25 degrees C and analyzed its molecular size using SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The results indicate that CTX forms a complex of approximately 145 kDa on the surface of erythrocytes and lipid vesicles, and that the complex formation is closely correlated with the toxin-induced permeabilization of target membranes. Thus, CTX may assemble into a pore-forming oligomer on target membranes.

Cytotoxin-converting phages, phi CTX and PS21, are R pyocin-related phages.

phi CTX is a temperate phage of Pseudomonas aeruginosa harbouring the ctx gene that encodes cytotoxin (CTX). We identified phi CTX as an R pyocin-related phage, by serological and molecular analysis, based on the findings that the infectivity of the phage was inhibited with the antisera directed R pyocins and R pyocin-related phages and that the phi CTX genome showed DNA homology to the genome of PS17 (a representative of the R pyocin-related phages) as well as to the pyocin R2 genes. Another new CTX-converting, R pyocin-related phage named PS21 was isolated from a CTX-producing strain of P. aeruginosa, suggesting the distribution of the ctx gene by certain members of R pyocin-related phage family.

Characteristics of antinociception induced by noncatecholic phenylethylamine derivatives: the involvement of alpha-2-adrenoceptors.

Characteristics of the antinociceptive action of phenylethylamine derivatives, amphetamine, beta-phenylethylamine (PEA) and beta-hydroxyphenylethylamine (OHPEA), were examined. The antinociception induced by PEA derivatives was enhanced by intracisternal injection of norepinephrine or clonidine and attenuated by intracisternal injection of phentolamine or yohimbine, but was not affected by intracisternal injection of prazosin in the mouse hot plate method. PEA derivatives induced a contraction of the rat vas deferens, and this contraction by PEA derivatives was attenuated by the application of phentolamine. The contractions induced by PEA or OHPEA in the reserpinized vas deferens were much smaller than those in the normal one. PEA derivatives inhibited the electrical stimulation-evoked contractions of the vas deferens, and the inhibition by PEA derivatives was reversed by the application of yohimbine. These findings indicate that PEA derivatives may induce the antinociception as a result of stimulating the alpha 2-adrenoceptors. The stimulation of alpha 2-adrenoceptors by PEA derivatives may result from the release of endogenous norepinephrine and/or from direct action on the alpha 2-adrenoceptors.

Molecular analysis of a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa: structure of the attP-cos-ctx region and integration into the serine tRNA gene.

The Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage phi CTX. The genome of phi CTX is a 35.5 kb double-stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of phi CTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP-cos-ctx region. The termini of phi CTX are 21-base 5' extended-single-stranded DNAs. The ctx gene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP-cos-ctx region contains six kinds of repeats and integration host factor-binding sequences and showed sequence-directed static bends, suggesting its potential to form a highly ordered structure. In addition, phi CTX was found to integrate into the serine tRNA gene which was mapped to the 43-45 min region on the P. aeruginosa chromosome.

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.