Performance of recombinant inbred lines in Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Performance of a random array of recombinant inbred lines derived by single seed descent from five different source populations of Brussels sprouts (Brassica oleracea var. gemmifera) is presented. A total of 2,356 lines were tested in trials during 1985 and 1986. Three of the source populations were derived from double crosses between F₁ hybrids. These hybrids show a considerable heterotic advantage over their inbred parents for the most important agronomic traits. The recombinant inbred lines performed, on average, less well than the parental inbred material, indicating that additive x additive genie interactions may make a significant contribution to the performance of current inbred material. Nevertheless, the very large variation among the recombinant inbred lines permitted many lines to be identified which outperformed the best parental inbred for all traits. Two lines outperformed the reference F₁ hybrid, Gower, for an index that included marketable yield and quality. Consideration was also given to the dangers of misinterpreting phenotypically based proportions. Accordingly, response equations were used to ascertain the real genetic progress that was made. Advance seemed small when compared with the large heterotic effect, which is consistent with the segregation of a large number of loci. The distribution of the recombinant inbred lines was compared to predictions made from early generation trials. There was broad agreement but significant discrepancies existed which, it is suggested, may arise from the effects of genotype-environment interactions.     

Method to estimate genotype probabilities at individual loci in farm livestock

Summary A Bayesian method to estimate genotype probabilities at a single locus using information on the individual and all its relatives and their mates has been developed. The method uses data over several generations, can deal with large numbers of individuals in large livestock families and allows for missing information. It can be extended to multiple alleles and can be used for autosomal or sex-linked loci. The allele frequencies and the form of expression (dominance, penetrance) must be specified. An algorithm using the method and involving an iterative procedure has been developed to calculate the genotype probabilities for practical use in livestock breeding. The method and algorithm were used to determine the accuracy of estimating genotype probabilities of sires for a female sex-limited trait, such as genetic variants of milk proteins. Data were similated and genotype probabilities estimated for 100 sires (20 replicates) with 3, 6 and 12 female offspring per sire, for different population frequencies, for additive and dominance gene action and for variable genotypic expression. Such simulation is useful in the design of testing systems for the use of information on specific genetic loci in selection.Prepared during a leave at Centre for Genetic Improvement of Livestock, Guelph, Canada     

Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Summary Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.     

A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.     

The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.     

Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro

The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.     

A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility

A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analyzed for the height (cm) and time (min) to the peak deflection. To standardize the procedure, a fixed number of cells (1x10(9)) were used to prepare suspensions of 300x10(6) cells/ml. Coefficients of variation for mean values obtained under these conditions after the evaluation of five ejaculates from a given stallion were estimated at between 10 and 12%. Correlations between swim-up measurements and computer-assisted semen analysis demonstrated that the percentage of motile cells and mean velocity (mum/sec) of motile cells influenced swim-up measurements. Described here is a simple and inexpensive procedure to determine objective measurements of spermatozoan motility that may have application in semen evaluation and fertility testing in the stallion.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.