Pigs expressing salivary phytase produce low-phosphorus manure.

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

Bombyx mori nuclear polyhedrosis virus-based vectors for expressing passenger genes in silkmoth cells under viral or cellular promoter control.

The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5'-untranslated or 5'-flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.

Comparative carcass and tissue nutrient composition of transgenic Yorkshire pigs expressing phytase in the saliva and conventional Yorkshire pigs.

A transgenic line of Yorkshire (YK) pigs named the Cassie (CA) line was produced with a low copy number phytase transgene inserted in the genome. The transgenic line efficiently digests P, Ca, and other major minerals of plant dietary origin. The objectives of this study were to 1) compare carcass and tissue nutrient composition and meat quality traits for third generation hemizygous CA line market BW finisher pigs (n = 24) with age-matched conventional YK finisher pigs (n = 24) and 2) examine effects of outbreeding with high-index conventional YK boars on modifying carcass leanness from the third to sixth generations in CA line finisher boars (n = 73) and gilts (n = 103). Cassie boars (n = 12) and CA gilts (n = 12) were fed diets without supplemental P and comparable numbers of age-matched YK boars and gilts fed diets containing supplement P were raised throughout the finisher phase. The pigs were slaughtered and then fabricated into commercial pork primals before meat composition and quality evaluation. Proximate and major micronutrient composition was determined on tissues including fat, kidney, lean, liver, and skin. The main difference observed was greater (P = 0.033) crude fat content in CA boar carcasses and increased (P < 0.04) leaf lard in both CA boars and gilts but no differences were observed (P = 0.895 and P = 0.223, respectively) in carcass backfat thickness as compared with YK pigs. There were no substantive differences in tissue composition, except for CA boar kidneys. Numerous changes in the mineral, fatty acid, and indispensable AA composition for CA boar kidneys were not apparent in CA gilts. These changes may point to adaptive physiological changes in the boar kidney necessary for homeostatic regulation of mineral retention related to phytase action rather than to insertion of the transgene. However, from a meat composition perspective, transgenic expression of phytase in the CA line of YK pigs had little overall effect on meat composition. Outbreeding of high-index CA gilts with high-index commercial YK boars linearly reduced (P = 0.002) back fat thickness with a corresponding linear increase (P = 0.001) in lean yield in finisher CA gilts, although no change in these parameters was observed in CA finisher boars. The increase in lean yield in CA gilts by selective breeding without affecting the level of salivary phytase activity documents the value of conventional genetic selection in conjunction with genetic modification.

Phytase properties and locations in tissues of transgenic pigs secreting phytase in the saliva.

A transgenic Cassie (CA) line of Yorkshire (YK) pigs was developed using a transgene composed of the mouse parotid secretory protein promoter linked to the Escherichia coli phytase gene integrated in chromosome 4. Previous studies documented that salivary secretion of phytase was sufficient to enable efficient digestion of plant feed phytate P. In the present study the catalytic properties and tissue distribution of the phytase in CA pigs were determined by a combination of enzymatic assays, immunohistochemistry, and immunoblots of tissue samples. The E. coli phytase had a mass of 44.82 kDa whereas the phytase secreted in CA saliva had a mass of 52.42 kDa as a result of glycosylation of the enzyme in the parotid gland. Despite the difference in size, the 2 enzymes exhibited similar substrate specificities, and substrate affinity ( K: m) and maximum hydrolytic activity ( V: max) catalytic properties. Phytase assays showed that the enzyme was present at high specific activity in the salivary glands with low activity in the soft palate and essentially none in the kidney, lean (muscle), liver, or skin of CA pigs and none in YK pigs. This conclusion was supported by immunoblot analysis using a polyclonal anti-phytase antibody. Immunohistochemical analysis of 83 different tissue locations of CA and YK pigs confirmed the ubiquitous presence of phytase in serous cells of the salivary glands and the localized presence of phytase in both serous and mixed cell types in the submucosal glands of the oropharynx; in the pharynx, tonsils, and esophagus; in some Bowman's glands in the nasal mucosa and eustachian tube; and in the prostate gland of CA boars. Furthermore, it showed the absence of phytase from the kidney, lean, liver, and skin of CA pigs. Phytase was not detected in any of the conventional YK tissues tested. The phytase was found to be glycosylated with the allergenic galactose-α-1,3-galactose (α-gal) epitope by immunoblotting using α-gal specific monoclonal antibodies. Galactose-α-1,3-galactose glycosylation of proteins is a common feature of pork and other red meats. The α-gal epitope was shown to be associated with a few proteins in muscle and skin but with the greatest number of proteins in kidney and parotid tissues of CA and YK pigs. The absence of phytase from the major food tissues and the displacement of other α-gal glycosylated proteins in the parotid glands by α-gal glycosylated phytase in conjunction with previously published data support the contention that expression of the novel phytase has minimal influence on pork quality and safety.

Digestive utilization of phosphorus from plant-based diets in the Cassie line of transgenic Yorkshire pigs that secrete phytase in the saliva.

A line of transgenic Yorkshire pigs referred to as the Cassie (CA) line was generated, which possessed a stable, low copy number phytase transgene insertion that enabled phytase secretion in the saliva. This study was conducted to assess growth and efficacy for improving P, Ca, and other macromineral utilization in the CA pigs receiving diets typical of those used for commercial swine production. In Exp. 1, 12 CA boars and 12 CA gilts fed diets without supplemental P gained weight and exhibited feed efficiency similar to conventional age-matched 12 Yorkshire boars and 12 Yorkshire gilts raised on similar diets with supplemental P. Serum concentrations of P and Ca were similar for CA and Yorkshire pigs during the growing and finishing phases, indicating that the CA pigs were not P limited. In Exp. 2, 6 CA (13.1 kg BW) and 6 Yorkshire barrows (8.8 kg BW) were fed 3 diets (control; low in Ca and P; and low in Ca, P, and CP) over 3 phases. The CA barrows fed the diet without supplemental P retained 25 to 40% (P < 0.001), 77 to 91% (P < 0.001), and 27 to 56% (P < 0.001) more P during the weaning, growing, and finishing phases, respectively, than conventional Yorkshire barrows fed similar diets without supplemental P. In Exp. 3, CA and Yorkshire barrows of similar ages weighing 66.2 ± 1.7 kg (n = 10) and 50.0 ± 1.0 kg (n = 10), respectively, were used. The P retention of CA finisher barrows fed a diet without supplemental P was 34% greater (P < 0.001) than conventional Yorkshire barrows fed the same diet with 750 units of exogenous phytase/kg diet. Urinary Ca to P ratio in the CA pigs was 0.27, whereas that for the Yorkshire barrows was 30, thereby, indicating that the Yorkshire barrows suffered a P deficiency. Furthermore, digestive utilization of major electrolyte macrominerals, K and Na, was improved (P < 0.05) by 18 and 16%, respectively, in the CA finisher pigs compared with the conventional Yorkshire finisher pigs fed phytase; however, only K exhibited enhanced retention. In conclusion, the CA line pigs secrete sufficient phytase from the salivary glands to enable efficient digestion of plant P, Ca, and major electrolyte macrominerals.

Genetic and developmental characterization of the aldox-2 locus of Drosophila melanogaster.

The aldox-2 locus in Drosophila melanogaster has been shown to affect differentially three molybdoenzymes, aldehyde oxidase, pyridoxal oxidase, and xanthine dehydrogenase. These effects are most obvious at times surrounding the pupal-adult boundary, when the normal organism accumulates large amounts of these enzymes in their active form. This locus has been more precisely mapped genetically to 2-82.9 +/- 2.1, with complete concordance between the effects of all recombinant chromosomes on all three enzymes. The cytogenetic location has also been determined to be between 52E and 54E8, with the likelihood that it lies within the region 54B1-54E8. The aldox-2 mutant allele has no visible phenotype and is completely recessive for enzyme effects at all stages tested. Segmental duplication of this region, including the aldox-2+ allele, has no apparent effect on the visible phenotype or the enzymatic activity. The mutant aldox-2 allele has no effect on the developmental expression of two unrelated enzymes, 6-phosphogluconate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. The effects of this locus on aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase suggest that this locus may code for a product involved in the synthesis of the molybdenum cofactor common to these enzymes.

Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection.

Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required.

The aldox-2 locus of Drosophila melanogaster also affects sulfite oxidase and molybdenum metabolism.

Mutation at the aldox-2 locus in Drosophila melanogaster affects the specific activities of four molybdoenzymes differentially during development. Sulfite oxidase activity is normal during late larval and pupal stages but is reduced during early adult stages in aldox-2 organisms. There was complete concordance among the effects of aldox-2 on sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase, when 38 stocks were analyzed which were derived from single recombination events between c and px, markers which flank aldox-2. Several different biochemical analyses indicate that the active molybdoenzymes present in the aldox-2 strain are normal with respect to size, shape, pH-activity profile, Km, and molecular weight. Significant differences were found between the aldox-2 strain and the OR control strain in their responses to dietary Na2MoO4 and Na2WO4. The mutant strain is much more resistant to the effects of dietary Na2WO4 and much more responsive to the administration of Na2MoO4 than the OR control strain when these effects are quantitated by measurements of molybdoenzyme specific activities. This evidence suggests that the aldox-2+ gene product has a molybdenum binding site which can also bind tungsten and that this site is altered in the mutant strain. The hypothesis presented explains the observed effects of the aldox-2 mutation and relates them to the other mutations reported in this gene-enzyme system.

A cellular promoter-based expression cassette for generating recombinant baculoviruses directing rapid expression of passenger genes in infected insects.

We have developed an expression cassette which allows the generation of recombinant baculoviruses that can express passenger genes under the control of a constitutive cellular promoter derived from the cytoplasmic actin gene of the silkmoth Bombyx mori. Silkmoth tissue culture cells which were infected with a recombinant B. mori nuclear polyhedrosis virus (BmNPV) containing the gene-encoding chloramphenicol acetyltransferase (CAT) under the control of this expression cassette expressed significant CAT activity beginning 5 hr postinfection (p.i.). Cells infected with a recombinant BmNPV containing the cat gene under the control of the polyhedrin gene promoter did not express CAT activity until 20 hr p.i. Silkworm larvae were also infected with the two recombinant viruses by hemocelic injections and all larval tissues examined were found to express the cat gene. While significant actin-cassette-driven CAT expression in vivo was first seen at 24 hr p.i., expression from the polyhedrin promoter was not seen until 48 hr p.i. By 60 hr p.i., tissues of larvae infected with the recombinant virus expressing cat under polyhedrin promoter control were found to exhibit sixfold higher CAT activity than those infected with recombinant virus expressing the cat gene under the control of the actin promoter. The 24-hr temporal advantage in expression of a passenger gene in infected larvae indicates that the actin-promoter-based expression cassette or other analogous cellular promoter-based cassettes could be used for generating recombinant baculovirus insecticides which could incapacitate pest insects more quickly than viruses employing the polyhedrin or other late viral promoters for expressing insect-incapacitating proteins.

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster.

We have characterized biochemical effects of IdhGB1 in Drosophila melanogaster. This is a "null"-activity allele for NADP+-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The Km values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP+ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.