Towards interbreed IBD fine mapping of the mh locus: Double-muscling in the Asturiana de los Valles breed involves the same locus as in the Belgian Blue cattle breed

The Spanish ``Asturiana' cattle breed is characterized by the segregation of a genetically determined muscular hypertrophy referred to as double-muscling or ``culones'. We demonstrate by linkage analysis that this muscular hypertrophy involves the mh locus previously shown to cause double-muscling in the Belgian Blue cattle breed, pointing towards locus homogeneity of this trait across both breeds. Moreover, using a twopoint and multipoint maximum likelihood approach, we show that flanking microsatellite markers are in linkage disequilibrium with the mh locus in both breeds albeit with different alleles. Finally, we discuss how allelic homogeneity across breeds might be exploited to achieve efficient genetic fine-mapping of the mh locus. Received: 13 September 1996 / Accepted: 20 January 1997     

The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution.

The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.     

Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.     

Limitation of DNA-4',6-diamidine-2-phenylindole assay in the presence of an excess of tRNA

4',6-Diamidino-2-phenylindole, DAPI, forms fluorescent complexes with DNA. This property has been used to quantify DNA on the basis of fluorometric test. However, the fluorescence quantum yield of DAPI increases also with tRNA. DNA estimation needs particular care in the presence of tRNA. For DNA containing 50% adenine-thymine (AT), DAPI can be used if DNA represents at least 3.4% of the total nucleotide material. This percentage varies with the AT/guanosine-cytosine content. When the fraction of DNA decreases further, the DAPI assay can no longer be used.     

Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.     

Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.