Collection and characterization of semen in Mithun (Bos frontalis) bulls

The objective of this study was to collect semen from semiwild Mithun (Bos frontalis) bulls using an artificial vagina (AV) and to determine semen characteristics. Collection of semen with an AV was attempted in five Mithun bulls using both anestrous and estrous Mithun females. No Mithun bull mounted an anestrous female Mithun during 60 trials, but satisfactory mounting, including extension of the penis, occurred in 25 trials with estrous Mithun females. In 15 of these trials, semen was successfully collected in an AV with an internal temperature of 42 to 46 °C. However, in 10 trials with an AV with an internal temperature of 36 to 40 °C, semen was not collected. Mean (± SEM) intervals to first mount and to ejaculation in the AV were 27.9 ± 3.6 sec and 113.8 ± 6.6 sec, respectively. Semen volume and pH were 3.1 ± 0.35 mL and 6.59 ± 0.04, and mean mass activity (scale, 0 to 4), initial sperm motility, live sperm count, sperm concentration, total number of sperm in the ejaculate, and overall sperm length were 2.2 ± 0.3, 78.6 ± 2.6%, 80.7 ± 2.2%, 710.8 ± 66.8 × 10⁶/mL, 2114 ± 364.4 sperm, and 67.9 ± 0.6 μm, respectively. The proportion of morphologically normal sperm was 80.6 ± 0.2%, whereas the proportion with a morphologically abnormal head, midpiece, tail, and acrosome were 4.2 ± 0.4%, 1.6 ± 0.5%, 6.1 ± 1.1%, and 7.1 ± 0.9%, respectively. The mean incidence of tail-less heads and proximal and distal protoplasmic droplets were 0.5 ± 0.1%, 0.3 ± 0.2%, and 2.4 ± 0.3%, respectively. In conclusion, we successfully collected semen from semiwild Mithun bulls with an AV maintained at 42 to 46 °C, and overall, the semen was within the normal range of that collected from fertile domestic bulls.     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.     

Isolation and characterization of ten microsatellite loci in Blue-footed (Sula nebouxii) and Peruvian Boobies (Sula variegata)

Ten microsatellite loci were isolated and characterized from Blue-footed (Sula nebouxii) and Peruvian Boobies (S. variegata). The loci were screened in 24 Blue-footed Boobies and 27 Peruvian Boobies: 8 were polymorphic in Blue-footed Boobies with between 2 and 10 alleles per locus and 9 were polymorphic in Peruvian Boobies with between 2 and 12 alleles per locus. Observed heterozygosity ranged from 0.29 to 0.84. These loci were also tested in Brown Boobies (S. leucogaster) and were variably polymorphic. These new loci are currently being used to assess population genetic structure in Blue-footed and Peruvian Boobies and will also be used to examine hybridization between the species.     

Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide

Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.     

Evolution of the alcohol dehydrogenase (ADH) genes in yeast: characterization of a fourth ADH in Kluyveromyces lactis

Summary Three alcohol dehydrogenase (ADH) genes have recently been characterized in the yeast Kluyveromyces lactis. We report on a fourth ADH in K. lactis (KADH II: KADH2 gene) which is highly similar to other ADHs in K. lactis and Saccharomyces cerevisiae. KADH II appears to be a cytoplasmic enzyme, and after expression of KADH2 in S. cerevisiae enzyme activity comigrated with a K. lactis ADH present in cells grown in glucose or in ethanol. KADH I was also expressed in S. cerevisiae and it comigrated with a major ADH species expressed under glucose growth conditions in K. lactis. The substrate specificities for KADH I and KADH II were shown to be more similar to that of SADH II than to SADH I. SADH I cannot efficiently utilize long chain alcohols, in contrast to other cytoplasmic yeast ADHs, presumably because of the presence of a methionine (residue 271) in its substrate binding cleft. A comparison of the DNA sequences of ADHs among K. lactis, S. cerevisiae and Schizosaccharomyces pombe suggests that the ancestral yeast species contained one cytoplasmic ADH. After divergence from S. pombe, the ADH in the ancestor to K. lactis and S. cerevisiae was duplicated, and one ADH became localized to the mitochondrion, presumably for the oxidative use of ethanol. Following the speciation of S. cerevisiae and K. lactis, the gene encoding the cytoplasmic ADH in S. cerevisiae duplicated, which resulted in the development of the SADH II protein as the primary oxidative enzyme in place of SADH III. In contrast, the K. lactis mitochondrial ADH duplicated to give rise to the highly expressed KADH3 and KADH4 genes, both of which may still play primary roles in oxidative metabolism. These data suggest that K. lactis and S. cerevisiae use different compartments for their metabolism of ethanol. Our results also indicate that the complex regulatory circuits controlling the glucose-repressible SADH2 in S. cerevisiae are a recent acquisition from regulatory networks used for the control of genes other than SADH2.

---

     

Identification of alkaline stress-responsive genes of CBL family in sweet sorghum (Sorghum bicolor L.)

Calcineurin B-like proteins play important roles in the calcium perception and signal transduction of abiotic stress. In this study, the bioinformatic analysis of molecular characteristics of Sorghum bicolor calcineurin B-like protein (SbCBL) revealed that sequences of SbCBL are highly conserved, and most SbCBLs have three typical EF-hands structures. Among the SbCBL proteins, four of which, SbCBL01, 04, 05, 08, have a conserved N-myristoylation domain. Stress-responsive and phytohormone-responsive cis-elements were found in the promoter regions of SbCBL genes. Real-time quantitative polymerase chain reaction (RTqPCR) analysis showed that SbCBL genes have different tissue-specific expression patterns under normal growth conditions in sweet sorghum (Sorghum bicolor L. Moench). Interestingly, when treated with sodium carbonate, SbCBL genes also show various sodium carbonate stress responsive patterns in sweet sorghum seedlings. These results suggest that SbCBLs may participate in regulating sodium carbonate stress-specific cellular adaptation responses and influencing growth and developmental patterns in sweet sorghum.     

Map position of Aldox (Aldehyde-oxidase) and Adh (Alcohol dehydrogenase) loci on autosome II of Musca domestica L.

The Aldox and Adh structural loci of Musca domestica L. belong to autosome II. They code for the enzymes aldehyde oxidase and alcohol dehydrogenase. Both these enzymes have allelic variants with specific electrophoretic mobility, which, on cellogel, are seen as single bands. The Aldox and Adh loci encompass a large map interval, which includes the morphological markers ar, cm, and car. The recombination frequencies between these five loci indicate the alignment Aldox-ar-cm-car-Adh.

---

Localisation chez Musca domestica L. des gènes Aldox et Adh codant les enzymes ald\;ehyde oxydase (AO) et alcool déshydrogénase (ADH)

Résumé Chez la mouche, les enzymes AO et ADH, observées par électrophorèse sur cellogel, présentent toutes 2 des formes alléliques exprimées par des bandes ayant une mobilité anodique propre.Les gènes structuraux Aldox et Adh, codant ces formes, sont liés entre eux et situés sur le chromosome 2. Ils se recombinent avec une fréquence élevée d'interchange; ils sont donc séparés par un intervalle important dans lequel sont compris les caractères visibles ar, cm, car. La fréquence des recombinaisons entre caractères visibles et gènes enzymatiques indique l'ordre suivant sur ce segment du deuxième chromosome de la mouche: Aldox, ar, cm, car, Adh.

     

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.Communicated by M.-A. Grandbastien     

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF^ts1. The mutations that resulted in a temperature-sensitive phenotype in mSF^ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 °C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF^ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF^ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger

The effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration. The optimum pH values for growth and metal ion accumulation were determined as 5.0, 4.5 and 3.5 for copper(II), lead(II) and chromium(VI) ions, respectively. Although all metal ion concentrations caused an inhibition effect on the growth of A. niger, it was capable of removing of copper(II) and lead(II) with a maximum specific uptake capacity of 15.6 and 34.4 mg g⁻¹ at 100 mg dm⁻³ initial copper(II) and lead(II) concentration, respectively. Growth of A. niger was highly effected by chromium(VI) ions and inhibited by 75 mg dm⁻³ initial chromium(VI) concentration since some inhibition occurred at lower concentrations.     

Isolation and characterization of microsatellite markers in the caddisfly Drusus discolor (Trichoptera: Limnephilidae)

We describe the development of and amplification conditions for microsatellite primers isolated from the caddisfly Drusus discolor. Eight polymorphic microsatellite loci were developed and screened for variability using 37 individuals from two populations from central Europe. The primers yielded an average of 8.6 alleles per loci. No linkage disequilibrium between loci was detected, while three loci showed deviations from Hardy–Weinberg equilibrium in one of the two tested populations.     

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30–40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans. Received: 26 November 1996 / Accepted: 30 January 1997     

Genetic and physical characterization of theLR1 leaf rust resistance locus in wheat (Triticum aestivum L.)

The objective of this study was to characterize the leaf rust resistance locusLr1 in wheat. Restriction fragment length polymorphism (RELP) analysis was performed on the resistant lineLr1/6^*Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F₂ populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism betweenLr1/6^*Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs)Lr1/6^*Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F₂ populations. One probe (pTAG621) showed very tight linkage toLr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing theLr1 resistance gene in different genetic backgrounds showed the same band asLr1/6^*Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant lineLr1/6^*Thatcher. This STS, specific for theLr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked toLr1 should ultimately provide the basis for positional cloning of the gene.