Royal A. McGraw

Microarray profiling of gene expression during trypomastigote to amastigote transition in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, remains a significant public health concern throughout South and Central America. Although much is known about immune control of T. cruzi and in particular the importance of recognition of parasite-infected cells, relatively little is known about the target antigens of these protective immune responses. For instance, few of the genes expressed in the intracellular amastigote stage have been identified. To gain insight into the molecular events, at the level of mRNA abundance, involved in this critical point in the parasite life-cycle, we used DNA microarrays of 4400 sequences from T. cruzi ORF-selected and random, genomic sequencing libraries to determine relative mRNA abundances in trypomastigotes and developing amastigotes. Results from six hybridizations using independently generated parasite samples consistently identified 60 probes that detected genes upregulated within 2h after extracellular trypomastigotes were induced, in vitro, to differentiate into amastigotes. Sequence analysis from these 60 probes identified 14 known and 25 novel T. cruzi genes. The general direction of regulation was confirmed by quantitative RT-PCR for seven of the array-identified, amastigote upregulated, known genes. This work demonstrates the feasibility of computational and microarray approaches to gene discovery in T. cruzi, an organism for which a fully assembled and annotated genome sequence is not yet available and in which control of transcription initiation is believed to be absent. Moreover, this work is the first report of amastigote up regulation for 38 genes, thus expanding considerably the pool of genes known to be upregulated in this important yet poorly-studied stage of the T. cruzi life-cycle.

Trichinella spiralis (T1) and Trichinella T5 : A comparison using animal infectivity and molecular biology techniques

We compared Trichinella T5 of bobcat (Lynx rufus) origin with Trichinella spiralis (T1) by using animal infectivity and molecular biology techniques. Swine, SD rats, and CF1 mice were highly resistant to infection with Trichinella T5 but sensitive to T. spiralis, whereas deer mice (peromyscus maniculatus) had similar sensitivity to both parasites. The fecundity of Trichinella T5 in deer mice was 10-35-fold higher in comparison to the fecundity in laboratory rodents (SD rats and CF1 mice). Fecundity of T. spiralis was approximately the same in both groups. A western blot, using excretory-secretory proteins (ESP) from first-stage larvae of T. spiralis as antigen, showed similar banding patterns in the pigs infected with either T. spiralis or Trichinella T5, however, the homologous reaction was stronger than the heterologous reaction. Antibodies were detectable in swine sera commencing 3 or 5 wk postinfection with T. spiralis or Trichinella T5, respectively. Complementary DNAs encoding the 46-, 49/43-, or 53-kDa ESP showed 3.54, 1.94, and 5.91% differences, respectively, between the 2 parasites. Deduced amino acid sequences of the 3 cDNAs were different at 7.20, 5.08, and 8.55%, respectively. All recombinant proteins of the 3 cDNAs from both parasites could detect antibodies in positive sera. The sequences of cDNAs encoding the 46-, 49/43-, or 53-kDa ESP from T. spiralis are also compared to the previously reported sequences, and the differences are discussed.

Cloning and characterization of gene TNF α encoding equine tumor necrosis factor alpha

Abstract We report the molecular cloning and nucleotide sequence of the equine gene encoding tumor necrosis factor α. The 2610-bp genomic sequence was derived from three overlapping polymerase chain reaction products.

Monoclonal Antibodies to Porcine Tumor Necrosis Factor Alpha: Development of an Enzyme-Linked Immunosorbent Assay

Five hybridomas (4F4, 14H1, 9B4, 6E10, and 8G7) secreting antibodies to porcine tumor necrosis factor alpha (PTNF-alpha) were obtained from one fusion. Four of the 5 monoclonal antibodies (Mab) recognized recombinant PTNF-alpha (rPTNF-alpha) on western blot and were able to neutralize both rPTNF-alpha and native (released by porcine macrophages) PTNF (nPTNF-alpha, only 4F4, 14H1, and 9B4 tested for the neutralization of nPTNF-alpha) in vitro. A sandwich enzyme-linked immunosorbent assay (ELISA) for PTNF-alpha was developed using Mab 4F4 and purified rabbit polyclonal antibodies against PTNF-alpha. The test detected PTNF-alpha concentrations as low as 400 pg/ml and did not cross react with native porcine TNF-beta, recombinant human TNF-alpha, recombinant mouse TNF-beta or native mouse TNF-alpha. The Mabs and the ELISA should be useful for assessing PTNF-alpha levels in swine serum during disease processes and possibly for alleviation of toxic effects of TNF-alpha.

cDNA cloning of canine common α gene and its co-expression with canine thyrotropin β gene in baculovirus expression system

Abstract The common α gene of the canine glycoprotein hormones was cloned, sequenced and co-expressed with the canine thyrotropin β (TSHβ) gene in the baculovirus expression system, and a bioactive recombinant canine TSH was purified. The canine common α gene was cloned from the total RNA extracted from the canine pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR) using primers that were designed based on the consensus sequences from other species. The resulting 476 bp PCR product is consisted of the full coding sequence for the 96 amino acid mature α subunit, and a sequence encoding a 24 amino acid signal peptide. Homology analysis with other species revealed that the canine common α subunit potentially contains five disulfide bonds and two oligosaccharide chains N-linked to Asn residues located at positions 56 and 82. For expression in the baculovirus expression system, the common α gene was cloned downstream of the p10 promoter of the pAcUW51 transfer vector, and the previously cloned canine TSHβ gene was inserted under the polyhedrin promoter of the same vector. The recombinant virus containing both α and β genes was generated and propagated before being used to transfect the Sf9 insect cells for expression. The medium from the Sf9 cultures, presumably containing canine TSH α and β in native heterodimer confirmation, exhibited TSH bioactivity as indicated in the cAMP stimulation assay in FRTL-5 cells. The expressed recombinant protein was purified from the culture medium with an affinity column that was coupled with IgG purified from the polyclonal antibodies generated against the partially purified native canine TSH.

Canine thyrotropin β-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells.

Abstract The gene encoding the mature β subunit of canine thyroid stimulating hormone (cTSHβ) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSHβ purified from E. coli were generated. The gene fragment that encodes mature TSHβ was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSHβ gene and an intron of 450 bp. The two exons, which encode the mature cTSHβ subunit, was joined together by an overlap PCR and was expressed in E. coli as 6×His-tagged protein. The purified recombinant cTSHβ with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSHβ and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common α gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5′ end of the cTSHβ gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine α and canine TSHβ in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.

A complementary DNA encoding an antigen from Trichinella spiralis muscle larvae and its analog from Trichinella T5 of bobcat origin: sequence, cloning and expressions.

Reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA encoding an excretory-secretory (ES) antigen with mol. wt 45-50 kDa by SDS-PAGE from T. spiralis muscle larvae. The PCR product was purified by electrophoresis and sequenced by thermal cycle sequencing with primer walking. The cDNA is 890 bp long and encodes a polypeptide of 255 amino acid (AA) residues. Using the same methods, we also recovered a corresponding cDNA from Trichinella T5, which is 891 bp long and encodes 255 AAs. Comparison of the 2 Trichinella species indicates approximately 2.6% and 2.4% differences between the 2 cDNA sequences and between the 2 deduced AA sequences, respectively.

Cloning and characterization of hamster fetal retinoic acid receptor isoforms

Summary Hamsters are routinely employed in toxicology evaluation, particularly for investigating the teratologic potential of chemicals. We have employed Syrian golden hamsters in retinoid-induced teratogenesis, mechanisms of which involve various retinoic acid receptor (RAR) isoforms. The purpose of this study was to clone and characterize different full-length hamster RAR isoforms. A 12-day old fetal hamster cDNA library was constructed and screened for RAR isoforms using human or mouse probes. Three full-length clones representing RARα, β, and γ were isolated, amplified and sequenced, and based on their homology to known mammalian isoforms were termed as hamster RARα variant, RARβ2 and RARγ2, respectively. The respective translated products for these clones were 430, 448 and 406 amino acids long. The clones were homologous to their human or mouse counterparts, although differences, particularly in the N-terminal region, were observed. These differences may represent differential splicing of exons controlled by two promoters for each isoform.