Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

The roles of vitamin A for cytoplasmic maturation of bovine oocytes

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.     

Mapping the quantitative trait loci (QTL) for body shape and conformation measurements on BTA1 in Japanese Black cattle

The detection and mapping of segregating quantitative trait loci (QTL) that influence withers height, hip height, hip width, body length, chest width, chest depth, shoulder width, lumbar width, thurl width, pin bone width, rump length, cannon circumference, chest girth, abdominal width and abdominal girth at weaning was conducted on chromosomal regions of bovine chromosome one. The QTL analysis was performed by genotyping half‐sib progeny of five Japanese Black sires using microsatellite DNA markers. Probability coefficients of inheriting allele 1 or 2 from the sire at specific chromosomal locations were computed. The phenotypic data of progeny were regressed on these probability coefficients in a within‐common‐parent regression analysis using a linear model that included fixed effects of sex, parity and season of birth, as well as age as a covariate. F‐statistics were calculated every 1 cM on a linkage map. Permutation tests of 10 000 iterations were conducted to obtain chromosome‐wide significance thresholds. A significant QTL for chest width was detected at 91 cM in family 3. The detection of this QTL boosts the prospects of implementing marker‐assisted selection for body conformation traits in Japanese Black beef cattle.     

Methane emission from stored dairy manure slurry and slurry after digestion by methane digester

To examine the effect of storage temperature on the emission of methane (CH₄) and carbon dioxide (CO₂) and on recovery of nutrients from raw dairy manure slurry (RS) and slurry digested in a methane digester (DS), both slurries were stored in closed 100 L steel tanks under psychrophilic conditions (5, 10, 15 and 20°C) for a 150 day period. As the storage temperature increased, total methane emission increased in both types of slurry. The amount of methane emitted per unit of volatile solids of the RS and DS was 0.19 L/g and 0.10 L/g, respectively. The respective carbon dioxide emissions were 0.20 L/g for RS and 0.12 L/g for DS at 20°C of storage temperature. At temperatures greater than 15°C, the methane concentration in the emitted gas remained more than 40% of the total gas. During the experimental period, in excess of 90% of the total Kjeldahl nitrogen in these slurries was recovered (91.4–93.7% for RS and 93.7–98.4% for DS) after storage, and ammonium nitrogen was recovered in excess of 100% (100.1–143.2% for RS and 106.7–143.2% for DS storage tanks) because of the mineralization of organic nitrogen in the influent. These results indicate that manure slurry characteristics and storage temperature have significant impacts on methane emission. It can be concluded that on typical farms located in northern Japan, methane emission from manure storage tanks during late fall, winter and early spring may be negligible, because of manure temperatures less than 10°C. During late spring, summer and early fall, methane emissions can be substantially reduced by using underground storage to maintain lower manure temperatures.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.     

在日本北海道Tokachi district, 马铃薯晚疫病菌占主导地位的基因型的变化及不同基因型在水平抗病品种‘Matilda''上产生不同病斑的研究

在Memuro城镇，位于日本北海道Tokachi地区中部，主要种植田间抗病品种Matida。1998年马铃薯晚疫病在此品种上发生较前几年早。为了了解Matilda品种抗性降低的原因，对1996－1999年Tokachi地区的晚疫病菌基因型的时间变化进行了研究及不同基因型间病菌产生病斑能力进行了比较。在1997年以前基因型JP－1在Tokachi地区占主导地位。在1997年基因型A首次在Tokachi地区被发现并且局限在Tokachi北部。在1998和1999年基因型A遍及整个Tokachi地区，1999年其比例上升至72％。用孢子囊悬浮液接种检测不同基因型病菌对Matilda品种的致病力结果发现，基因型A比基因型JP－1和基因型B产生更大的病斑。然而通过液滴接种所有3个基因型都可产生扩展的病斑。结果表明，Matilda减少的田间抗性是由于基因型A传播到Memuro城镇的Matilda品种上所致。     

Seasonal changes in otolith and somatic growth in age-0 Pacific saury <Emphasis Type="Italic">Cololabis saira</Emphasis>

We evaluated the seasonal changes in otolith and somatic growth of age-0 Pacific saury Cololabis saira in 223 fish collected between June and November 2002. We calculated the age in days of each individual by measuring otolith growth increments under a scanning electron microscope. The age was correlated with body length and otolith radius. We also observed seasonal changes in the rate of increase in body length and otolith radius and in the pattern of otolith growth. Until August, both body length and otolith radius increased with age. Thereafter, the otolith radius continued to increase, whereas the rate of somatic growth decreased. As a result, the ratio of otolith radius to body length increased. After August, the percentage of otoliths with unreadable increments on their edge increased due to the formation of hyaline zones. Otoliths grew both radially and in thickness until July, but gradually stopped growing in thickness after August. Beginning in October, more than 80% of otoliths only grew radially. After August, the otolith not only continued growing but the morphological growth pattern also changed.