In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters

The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.     

The methylation state of 2 muscle-specific genes: restriction enzyme analysis did not detect a correlation with expression

To examine the possible role of DNA methylation in the modulation of expression of genes involved in the differentiation of muscle cells, we compared the methylation state of a number of CpG sites in the rat skeletal muscle actin and myosin light chain 2 genes, in muscle and nonmuscle cells, and in proliferating myoblasts and differentiated myotubes of the myogenic cell line L8. No correlation was detected between the state of methylation of these sites and the expression of the two genes. Essentially the same pattern of DNA methylation was observed, in the sites examined, in DNA from muscle, kidney and stomach. In DNA extracted from cultures of proliferating mononucleated myoblasts, as well as from differentiated multinucleated fibers of the myogenic cell line L8, the two genes were more methylated than in other tissues.     

The nucleotide sequence of the rat cytoplasmic beta-actin gene.

The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other actin genes, the beta-actin gene lacks the codon for Cys between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.     

Elimination of the disulphide bond alters the conformation of mature lipo-β-lactamase in yeast

Summary When expressed in Saccharomyces cerevisiae the precursor of the hybrid prokaryotic protein lipo--lactamase is accumulated in reduced form whereas the majority of the mature form contains an intra molecular disulphide bond (oxidized form). We have previously shown that mature mutant lipo--lactamases in which the cysteine residue 131 was changed to either tyrosine or threonine lack the capacity to form a disulphide bond but are nevertheless processed and secreted efficiently in yeast. Here we show that these mutant mature lipo--lactamases, in yeast cell extracts, exhibit a tenfold lower -lactamase-specific activity than the wild-type protein and that the mature mutant proteins are more susceptible to trypsin digestion. Thus, elimination of the disulphide bond alters the conformation of mature lipo--lactamase in yeast.Correspondence to: O. Pines     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

Multiple genotype analysis and sexing of IVF bovine embryos

Twenty-one in vitro-fertilized bovine blastocysts were quartered, lysed and subjected to primer elongation preamplification (PEP) procedure, allowing for the analysis of up to 40 genotypes per quarter embryo. The quarter-embryos were sexed by polymerase chain reaction (PCR) using BRY.1, Bov97M and ZFX/ZFY loci, and then genotyped for k-casein, bovine leukocyte adhesion deficiency (BLAD) and microsatellite D9S1. The mitochondrial cytochrome B locus was used as an internal control with a 95% success rate. The PEP procedure amplified genomic fragments in 93% of all cases. The embryos were identified to be 11 males and 10 females. Sexing accuracy was 87% for BRY.1, 97% for ZFX/ZFY and 100% for Bov97M. False genotyping was due mostly to amplification of BRY.1 in the female embryos and to the nonamplification of the ZFY locus in the male embryos. The results indicate that the combined use of Bov97M and ZFX/ZFY loci is a highly accurate procedure for sexing bovine embryos. Genotyping for kappa-casein, D9S1 and BLAD was successful in 94, 99 and 91% of assays, respectively. Sex ratios and allele frequencies of embryos for gk-casein, BLAD and D9S1 were all close to the observed frequencies in the Israeli Holstein population. These results support the conclusion that the genotyping of embryos is as accurate as that of mature animals. Thus, marker-assisted selection can be efficiently applied at the preimplantation embryo level for loci of economic importance.     

Does saline water consumption affect feeding and fuel deposition rate of a staging,long‐distance migrating passerine?

To accomplish their enduring journeys, migrating birds accumulate fuel consisting mainly of lipids in stopover sites located throughout their migration routes. Fuel deposition rate (FDR) is considered a key parameter determining the speed of migration and thereby bird fitness, and recent studies have demonstrated the positive effects of fresh water consumption on the FDR of migrating blackcaps Sylvia atricapilla. Sewage water reservoirs, characterized by higher water salinity than fresh water, were extensively built in different parts of the world and are used by birds during their travel, but their effects on wildlife and specifically on migrating birds have been largely overlooked thus far. We experimentally examined the effects of water salinity on blackcap FDR during migration. We captured birds in an autumn stopover site, transported them to the laboratory and provided them with fruits, mealworms and water of different salinity levels (0.3, 4.5 and 9‰ NaCl) for several days. We examined the effects of water salinity on the blackcaps’ diet, water consumption and FDR and found that FDR was mainly affected by fruit consumption rate and not by the water salinity levels. Water salinity nevertheless caused elevated water consumption as the birds consumed almost 3 times more saline water than fresh water per consumed fruit mass. Our work is the first to explore the consequences of saline water consumption on migrating passerines, specifically suggesting that anthropogenic alterations of habitats by sewage water treatment facilities may modulate bird nutrition and diet.