C. D. K. Bottema

Multiple functions for sterols in Saccharomyces cerevisiae.

Analyses with a yeast sterol auxotroph indicated that there are at least four different levels of function for sterol which have been designated sparking, critical domain, domain and bulk. Growth of yeast sterol auxotrophs on cholestanol is precluded unless minute amounts of ergosterol are available. We have designated this phenomenon the sparking of growth, in which cholestanol satisfies an overall membrane sterol requirement and ergosterol fulfills a high specificity sparking function. The critical domain role for sterol is observed under conditions of lanosterol supplementation where low levels of ergosterol (10-times those necessary for sparking on cholestanol) are required for growth. The sterol functions designated domain and bulk are illustrated by assessing cellular free sterol levels and plasma membrane properties of a sterol auxotroph after growth on different concentrations of exogenously supplied sterol. Plasma membranes isolated from auxotrophs grown on domain or bulk levels of sterol underwent no lipid thermotropic transitions, while plasma membranes from cells grown on critical domain levels of sterol underwent a lipid thermotropic transition, when analyzed by steady-state fluorescence anisotropy.

Gene structure alternative splicing, and chromosomal localization of pro-apoptotic Bcl-2 relative Bim.

Abstract. Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5′ to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases.

Fatty acid synthase effects on bovine adipose fat and milk fat.

A quantitative trait locus (QTL) was identified by linkage analysis on bovine Chromosome 19 that affects the fatty acid, myristic acid (C14:0), in subcutaneous adipose tissue of pasture-fed beef cattle (99% level: experiment-wise significance). The QTL was also shown to have significant effects on ten fatty acids in the milk fat of pasture-fed dairy cattle. A positional candidate gene for this QTL was identified as fatty acid synthase (FASN), which is a multifunctional enzyme with a central role in the metabolism of lipids. Five single nucleotide polymorphisms (SNPs) were identified in the bovine FASN gene, and animals were genotyped for FASN SNPs in three different cattle resource populations. Linkage and association mapping results using these SNPs were consistent with FASN being the gene underlying the QTL. SNP substitution effects for C14:0 percentage were found to have an effect in the opposite direction in adipose fat to that in milk fat. It is concluded that SNPs in the bovine FASN gene are associated with variation in the fatty acid composition of adipose fat and milk fat.

A comparison of the fatty acid composition of triacylglycerols in adipose tissue from Limousin and Jersey cattle

The fatty acid composition of the triacylglycerol fraction of shoulder fat from Limousin and Jersey yearling heifers, yearling steers, and non-lactating cows was investigated. Significant breed differences in the degree of fatty acid saturation were apparent between Jersey and Limousin cows, but were not observed in the yearlings. Jersey cows had less saturated fatty acids than the Limousin. Jersey cows showed an increased percentage of monounsaturated fatty acids compared with the Jersey yearlings. In contrast, the level of monounsaturated fatty acids in the Limousin cows was the same as in the Limousin yearlings. The calculated indices of enzyme activities also differed between the breeds. Jersey cows had higher indices of Δ9-desaturase and elongase activities than Limousin. This was also reflected by differences in the ratios of total unsaturated and polyunsaturated to saturated fatty acids. Breed differences were also observed in the triacylglycerol fatty acid chain length. In this case, however, yearlings showed significant breed differences that were not detected in the cows. Limousin yearlings had more long chain fatty acids (C16 and C18) than the Jersey yearlings. Limousin yearlings also had a higher elongase activity index than their Jersey counterparts. Thus, breed and age affect the fatty acid composition in these cattle.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

[18] Yeast sterols: Yeast mutants as tools for the study of sterol metabolism

Yeast mutants defective in ergosterol synthesis are valuable tools for investigating sterol metabolism. Both sterol mutants and sterol auxotrophs have been utilized in determining what sterol structural features are required for yeast cell viability. Both types of mutants can also be studied to ascertain how changes in sterol structure affect membrane properties. Other aspects of sterol metabolism, such as the specificity of sterol esterification, have been elucidated by the sterol auxotrophs. In broader applications, interrelationships between sterol metabolism and other cellular functions (e.g., heme metabolism) may also be examined with these mutants. By analyzing the lipid composition of the sterol mutants, on the other hand, much of the ergosterol biosynthetic pathway has been delineated. The unusual sterols of the mutants can also be obtained to develop assays for the enzymes involved in ergosterol synthesis. Thus, by utilizing mutants, the simple eukaryotic system of yeast may be extended to explore the entire field of sterol metabolism and its relationship to cellular physiology.

Polymerase Chain Reaction Amplification of Specific Alleles: A General Method of Detection of Mutations, Polymorphisms, and Haplotypes

Publisher Summary The polymerase chain reaction (PCR) can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in a method called “PCR amplification of specific alleles (PASA).” PASA has been further modified to include two allele-specific primers in the same PCR reaction. By varying the lengths of the allele-specific oligonucleotides, the amplified products can be distinguished from each other by gel electrophoresis. This method, called PCR amplification of multiple specific alleles (PAMSA), allows the rapid detection of more than one allele in a single PCR reaction. PASA is a generally applicable technique for the detection of point mutations or polymorphisms. The method may also be used to detect the presence of small deletions or insertions. PASA has the advantages of being rapid, reproducible, nonisotopic, and amenable to automation.

Effects of the myostatin F94L substitution on beef traits

This study investigated the effects of a SNP in the myostatin gene (MSTN or growth differentiation factor 8, GDF8) on birth, growth, carcass, and beef quality traits in Australia (Aust.) and New Zealand (NZ). The SNP is a cytosine to adenine transversion in exon 1, causing an amino acid substitution of leucine for phenylalanine(94) (F94L). The experiment used crosses between the Jersey and Limousin breeds, with the design being a backcross using first-cross bulls of Jersey x Limousin or Limousin x Jersey breeding, mated to Jersey and Limousin cows. Progeny were genotyped for the myostatin SNP and phenotyped in Aust., with finishing on feedlot (366 calves, over 3 birth years) and in NZ with finishing on pasture (416 calves, over 2 birth years). The effect of the F94L allele (A allele) on birth and growth traits was not significant. The F94L allele in Limousin backcross calves was associated with an increase in meat weight (7.3 and 5.9% of the trait mean in Aust. and NZ, respectively, P < 0.001), and a reduction in fat depth (-13.9 and -18.7% of the trait means on live calves (600 d) and carcasses, respectively, Aust. only, P < 0.001), intramuscular fat content (-8.2% of the trait mean in Aust., P < 0.05; -7.1% in NZ, not significant), total carcass fat weight (-16.5 and -8.1% of the trait mean, Aust. and NZ; P < 0.001 and P < 0.05, respectively). Meat tenderness, pH, and cooking loss of the M. longissimus dorsi were not affected by the F94L variant. In the Jersey backcross calves, additive and dominance effects were confounded because the F94L allele was not segregating in the Jersey dams. The combined effects, however, were significant on LM area (4.4% in both Aust., P < 0.05, and NZ, P < 0.01), channel fat (-11.7%, NZ only, P < 0.01), rib fat depth (-11.2%, NZ only, P < 0.05), and carcass fat weight (-7.1%, NZ only, P < 0.05). The results provide strong evidence that this myostatin F94L variant provides an intermediate and more useful phenotype than the more severe double-muscling phenotype caused by knockout mutations in the myostatin gene.

Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues.

Abstract The Eph family of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and “native” mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus. kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

Evidence that descendants of three founders constitute about 25% of hemophilia B in the united states

In our sample of 160 consecutive Caucasian hemophiliacs, 14 (9%) had a G----A transition at bp 10,430 that substitutes serine for glycine 60 in the first EGF domain of the factor IX molecule. Each of these hemophiliacs had clinically mild disease. Haplotype data and familial pedigrees indicate that 12 of these hemophiliacs are likely to be related to a common ancestor. The 13th and 14th patients possess different haplotypes and thus represent independent origins of the mutation. In addition, we have screened these 160 hemophiliacs for the previously reported mutations resulting from founder effects at IIe397----Thr and Thr296----Met. Herein we report an additional nine hemophiliacs with the mutant Thr397 allele and five additional hemophiliacs with the mutant Met296 allele. Haplotype data for these 14 hemophiliacs support the original founder effect hypotheses for these mutations. In total, the above three mutations are found in 44 of the 160 seemingly unrelated Caucasian hemophiliacs (28%). The sample includes patients from all regions of the United States and Ontario, Canada. Descendants of these three founders comprise approximately two-thirds of the missense mutations found in our sample of Caucasian hemophiliacs with clinically mild disease.