Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall.

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeating labeling experiments indicated that approximately 40% of these proteins were exposed on the extra-cytoplasmic surface of the plasma membranes of these cells. The studies demonstrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Assessment of the immunological and biological efficacy of two different doses of a recombinant GnRH vaccine in domestic male and female cats (Felis catus).

This study has assessed the immunological and biological efficacy of two different doses of a recombinant GnRH vaccine administered to intact domestic cats. Fifteen kittens, 8-9 weeks of age, were allocated to three unequal treatment groups: group 1, 1 male control cat; group 2, 5 females administered 10 microg of GnRH antigen; and group 3, 4 males and 5 females administered 100 microg of GnRH antigen. Animals in groups 2 and 3 were immunized at study days 0, 28 and 643. One of the four males (AJZ3) in the high dose group showed a more rapid decline in GnRH antibody titers and received an additional immunization at day 461. Blood samples were collected on study days 28, 35, 56, 97, 157, 213, 270, 325, 377, 433, 496, 549, 605, and 685. The injection sites were monitored for tissue reactivity on study days 5, 7, 12, and 28. The animals' general health and demeanor was monitored on a daily basis. Sera obtained from 11 animals on day 549 were submitted for biochemistry analysis. Two males and two females were necropsied at the completion of the study and histopathological examination of the gonads, hypothalamus, pituitary, kidneys and uterus was performed. All 14 immunized animals developed immunoneutralizing titers to GnRH. GnRH titers peaked at day 56 and 13 of 14 cats maintained these titers for >20 months. Except for AJZ3, the immunized males' serum testosterone concentrations were below the assay's level of detection after the second immunization. None of the 10 immunized females showed signs of estrous behavior or became pregnant. Testicular and ovarian histology was consistent with suppression of LH and FSH activity. The majority of tissue reactions resolved by 28 days post-vaccination. Serum biochemistry and tissue histopathology revealed no evidence of tissue or organ damage. This study was unique in that a recombinant GnRH antigen was used to stimulate and maintain biologically relevant titers in very young male and female cats for at least 20 months.

Hygromycin A, an antitreponemal substance. II. Therapeutic effect for swine dysentery.

This study was conducted to evaluate hygromycin A fed to growing swine at 1, 5, 10 or 20 g/ton feed for the control of Treponema hyodysenteriae-caused dysentery. Pigs provided carbadox at 50 g/ton feed served as an infected treatment control group. All pigs were orally, via stomach intubation, administered 100 ml of a T. hyodysenteriae broth culture. During the in vivo test, rectal swabs were taken for T. hyodysenteriae isolation, body weights of all pigs and the feed consumption was determined. All pigs were euthanized and necropsied at study end; the large intestine was cultured for T. hyodysenteriae and gross intestinal lesions were noted. T. hyodysenteriae-caused swine dysentery was successfully controlled by feeding hygromycin A at 5 g/ton. Hygromycin A medicated pigs performed as well as or better than carbadox-medicated pigs.

Pancreatic chymotrypsin as the essential enzyme for excystation of Eimeria tenella.

Sporocysts from the protozoan parasite, Eimeria tenella, were isolated, preincubated with sodium taurocholate, and treated with various preparations of pancreatic enzymes. Crude trypsin, crude lipase, and purified alpha-chymotrypsin all could break the shells of sporocysts and release sporozoites. Purified trypsin was much less active than crude trypsin and purified lipase showed no activity at all. Specific inhibitors of chymotrypsin, tosyl-L-phenylalanyl chloromethane, diphenylcarbamyl chloride, and chymostatin inhibited the release of sporozoites by all the enzyme samples, whereas tosyl-L-lysyl chloromethane, a specific inhibitor of trypsin, exerted no inhibitory effect. It is thus postulated that chymotrypsin, not trypsin, is an essential enzyme involved in excystation of E. tenella. Purified chymotrypsin is recommended to replace crude trypsin in the vitro excystation of E. tenella as a likely improved procedure.

Preparation and purification of merozoites of Eimeria tenella.

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.

Structure and composition of the oocyst wall of Eimeria tenella.

Oocyst walls were purified from unsporulated oocysts of Eimeria tenella. Analysis of the purified material indicated a composition of 67% peptide, 14% lipid, and 19% carbohydrate. The likely physical arrangement of the components places the lipid in a 10 nm thick outer layer, covering a 90 nm thick layer of glycoprotein. The protein component of the structure was dissociated using thiol reagents under denaturing conditions, and was shown to consist of a repeating subunit of approximately 10,000 daltons. The results suggest an explanation for the physical and mechanical resistance of the oocyst wall, as well as possible mechanisms for excystation of sporulated oocysts.

Precision genetics for complex objectives in animal agriculture.

Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.

Analysis of globin genes from murine erythroleukemia cells.

Globin structural genes from a murine erythroleukemia cell line were analyzed by Southern blot hybridization of genomic DNA and after isolation of cloned globin genes from a genomic library. The globin genes isolated from the erythroid cell line did not differ, when analyzed by extensive restriction endonuclease digestion, from globin genes isolated from nonerythroid cells. No gross structural differences were seen between murine erythroleukemia globin genes, either before or after hexamethylene bisacetamide (HMBA)-mediated erythroid differentiation, and globin genes from normal mouse liver DNA. Whereas the murine erythroleukemia genome hybridizes extensively to cloned Friend leukemia virus probes, there was no evidence of viral integration into sequences adjacent to the globin genes.

Dihydrofolate reductase from Eimeria tenella: rationalization of chemotherapeutic efficacy of pyrimethamine.

Dihydrofolate reductase activity of 0.2 nmole of dihydrofolate reduced/min/mg protein was detected in crude extracts of unsporulated oocysts of Eimeria tenella. The enzyme was purified by a combination of affinity and ion-exchange chromatography. Its molecular weight was estimated as 240,000 daltons. An anticoccidial drug pyrimethamine is a potent inhibitor of the activity of E. tenella dihydrofolate reductase (Ki = 3 nM), but it is less effective an inhibitor of dihydrofolate reductase from chicken liver. This difference may explain the in vivo therapeutic action of pyrimethamine against Coccidia.

Studies of a glycoprotein in the oocysts of Eimeria tenella.

A glycoprotein unique to the cytoplasm of the unsporulated oocyst of Eimeria tenella has been purified and partially characterized. The protein has a molecular weight of 30,000, of which approximately 40% is carbohydrate. The carbohydrate portion of the molecule consists of glucose, galactose, mannose, xylose, glucosamine, and galactosamine, with no detectable sialic acid. The protein portion contains approximately 141 residues, being rich in hydrophilic amino acids with very few aromatic amino acids and no cystine. The protein comprises 14% of the total soluble protein of the unsporulated oocyst but has not been identified in the cytoplasm of any other developmental stage of the organism. Using polyacrylamide gel electrophoresis and a radioimmunoassay specific for the protein, it has been shown to disappear from the cytoplasm between the 15th and 20th hour of the 20-hour sporulation process. Subsequent immunofluorescence experiments have shown a reactive material as a component of the sporozoite membrane. These results indicate that the glycoprotein is a structural protein of the sporozoite membrane, apparently synthesized by the unsporulated oocyst and incorporated into the sporozoite membrane as one of the last steps involved in the sporulation process.

Multiplicity of glutathione S-transferase genes in the rat and association with a type 2 Alu repetitive element.

Southern blot analysis of rat genomic DNA using glutathione S-transferase Ya and Yc cDNA probes was employed to estimate the size of the Ya/Yc multigene family. A minimum of five to seven Ya/Yc genes were detected; at least two of these are Yc genes. The presence of multiple genes was further supported by the isolation of three nonoverlapping genomic clones from a rat EcoRI library that hybridized to a Ya cDNA clone, pGTB38. However, not all EcoRI bands seen in genomic blots were represented in the clones, suggesting that not all Ya/Yc genes have been isolated. The organization of a Ya gene in one of these EcoRI genomic clones, lambda GTB38-3, and an overlapping clone, lambda GTB45-1, isolated from a HaeIII library, was investigated with 5' and 3' probes prepared from Ya and Yc cDNA clones. Restriction endonuclease mapping and hybridization studies revealed that the gene spans over 10 kilobases and contains at least three introns. Sequences upstream from the 5' untranslated region of the gene, and within an intron in the 5' coding region, were found to contain sequences homologous to a type 2 Alu repetitive element from the rat growth hormone gene [Page, G.S., Smith, S., & Goodman, H.M. (1981) Nucleic Acids Res. 9, 2087-2104]. The repetitive sequences in lambda GTB38-3 were identified by hybridization to a novel Ya cDNA clone, pGTB45. This cDNA clone was isolated from a cDNA library previously described [Telakowski-Hopkins, C.A., Rodkey, J.A., Bennett, C.D., Lu, A.Y.H., & Pickett, C.B. (1985) J. Biol. Chem. 260, 5820-5825] with nick-translated intron sequences as probes. pGTB45 is virtually identical with pGTR261 [Tu, C.-P.D., Lai, H.-C.J., Li, N.-Q., Weiss, M.J., & Reddy, C.C. (1984) J. Biol. Chem. 259, 9434-9439], except that the 3' untranslated region extends 231 base pairs beyond the polyadenylation signal of pGTR261. This elongated 3' untranslated sequence is unique in that it contains a full-length type 2 Alu repetitive element, which includes two additional, overlapping polyadenylation signals.