Complementary Deoxyribonucleic Acid Cloning Research Articles

Expression Profile of Two HSP70 Chaperone Proteins in Response to Extreme Thermal Acclimation inXestia c-nigrum(Lepidoptera: Noctuidae)

Heat shock proteins 70 (HSP70s) are highly evolutionarily conserved and play important roles in helping insects survive under extreme temperatures. In this study, Xestia c-nigrum (L.) (Lepidoptera: Noctuidae) was stressed at various temperatures, and the impacts on thermotolerance and cold endurance were examined. Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70), named Xc-HSC70 and Xc-HSP70, respectively, were isolated from X. c-nigrum using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Amino acid sequence analysis indicated that Xc-HSC70 and Xc-HSP70 contained the signature sequences of the HSP70 family, and phylogenetic analysis showed that Xc-HSP70s were consistent with the known insect classification. Genomic DNA structure analysis revealed that the Xc-HSC70 gene contained 8 introns that all conformed to the GT/AG rule, whereas the Xc-HSP70 gene did not contain any introns in the coding region. Fluorescent real-time quantitative RT-PCR revealed that both genes were significantly up-regulated under thermal stress and cold stress. The expression patterns indicated that Xc-HSP70 was heat inducible and Xc-HSC70 was constitutively expressed. The temperature response was more intense for Xc-HSP70 than for Xc-HSC70, regardless of the temperature or developmental stage; however, the expression of Xc-HSC70 and Xc-HSP70 was not altered in the pupa. Taken together, the expression profiles of both genes (Xc-HSP70s) provide good support to the insect for coping with adverse conditions.

Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant's schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5% identity and Sp-HSC70 shared more than 81.1% identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant's schizothoracin.

Complementary deoxyribonucleic acid cloning of avian G0/G1 switch gene 2, and its expression and association with production traits in chicken

As a member of the G0/G1 switch genes, G0/G1 switch gene 2 (G0S2) is related to many regulatory processes in the human and mouse. For example, it interacts directly with adipose triglyceride lipase to active its triglyceride hydrolysis activities. In this study, G0S2 gene cDNA of the chicken (522 bp), zebra finch (420 bp), sparrow (417 bp), pigeon (417 bp), and Bengalese finch (416 bp) were cloned, and each of them was encoded as a protein of 99 amino acids. The expression of G0S2 mRNA was determined by real-time reverse-transcription PCR analysis in 20 tested tissues of 21- and 91-d-old chickens. The highest mRNA level was found in abdominal fat and subcutaneous fat in both stages. Considerable G0S2 mRNA was also observed in chicken heart and muscle tissues. Expression of the chicken G0S2 gene varied at different stages and sexes. The abundance of G0S2 mRNA on d 21 was far higher than that on d 91. The abundance in female chickens was higher than that in males at both stages. In the coding region, we found 4 SNP, among which only G197A led to a change in the amino acids (Arg66Gln); the rest were synonymous substitutions. Association analysis showed that both G102A and G255A were significantly associated with head width (P < 0.05) and were highly significantly associated with leg muscle color (P < 0.01). The G102A was significantly associated with shank diameter at 63 d (P < 0.05). The SNP G197A was significantly associated with shank diameter at 49 d; CP content of leg muscle; total weights of the heart, liver, gizzard, and glandular stomach; and small intestine length (P < 0.05). In conclusion, much higher G0S2 mRNA was detected in both male and female chickens at 21 d of age than at 91 d of age, and 3 SNP (G102A, G197, and G255A) were associated with chicken production traits.

Overexpression of SlCZFP1, a Novel TFIIIA-type Zinc Finger Protein from Tomato, Confers Enhanced Cold Tolerance in Transgenic Arabidopsis and Rice

The C2H2-type (also called TFIIIA-type) zinc finger protein family is a major family of eukaryotic transcription factors. C2H2-type zinc finger proteins in plants play important roles in regulating various developmental processes or responses to abiotic stresses. We isolated a complementary deoxyribonucleic acid (cDNA) clone, designated Solanumlycopersicumcold zinc finger protein 1 (SlCZFP1), from tomato by analyzing the previous microarray data of the low-temperature transcriptome of tomato. SlCZFP1 encodes a novel TFIIIA-type zinc finger protein, which contains two typical zinc finger motifs, CX2–4 CX3FX3QALGGHX3–5H, and a potential nuclear localization signal (NLS). The SlCZFP1–GFP fusion protein was localized to the nucleus in a transient expression assay. Expression of SlCZFP1 was strongly induced by cold stress, dehydration, and salt treatment, but not by abscisic acid (ABA). Overexpression of SlCZFP1 in transgenic Arabidopsis and rice induced constitutive expression of cold-regulated (COR) or cold-responsive genes and conferred enhanced tolerance to freezing or cold treatments for non-acclimate transgenic plants, compared with wild-type plants. However, there was no obvious enhancement observed in drought and salt tolerance. Our data suggest that SlCZFP1 plays an important role in plant responses to cold stress by regulating cold-responsive gene expression and that SlCZEP1 might be a useful gene for improving cold tolerance in crop plants.

Molecular cloning and expression of two HSP70 genes in the Wuchang bream ( Megalobrama amblycephala Yih)

Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream ( Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were α-helix and random coil. Fluorescent real-time quantitative RT-PCR was used to study the effects of heat shock (34 °C), crowding stress (100 g L −1) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.

Cloning of adipose triglyceride lipase complementary deoxyribonucleic acid in poultry and expression of adipose triglyceride lipase during development of adipose in chickens

Increasing the breakdown of stored fat in adipose tissue leads to reducing fat content, enhancing feed efficiency and, consequently, decreasing the production cost of poultry. The processes of lipolysis are not completely understood, and the proteins involved in this process need to be identified. An adipose triglyceride lipase (ATGL), recently identified in several species, has not been studied in avian species. We have cloned the full-length coding sequences of ATGL cDNA for the chicken, turkey, and quail. Sequence comparisons among mammals and these avian species showed that the avian ATGL have 2 conserved domains, the patatin domain and the hydrophobic domain. The patatin domain contains lipase activity, and the hydrophobic domain exhibits lipid droplet binding. The high levels of chicken, turkey, and quail ATGL mRNA and protein are exclusively found in subcutaneous and abdominal adipose tissues. In addition, chicken ATGL (gATGL) is mainly expressed in the fractionated adipocytes compared with stromal-vascular cells that mostly contain preadipocytes (P < 0.001). Furthermore, ontogeny of gATGL mRNA and protein expression in adipose tissue showed induction of gATGL immediately after hatching before access to food (P < 0.05), suggesting that an energy deficit due to posthatching starvation may increase breakdown of stored fat via increasing gATGL expression in adipose tissue. Our studies showed that expression of the chicken ATGL is adipose specific and regulated developmentally, suggesting that a possible modulation of ATGL expression would regulate fat deposition in avian species.

Identification of Tropomyosins as Major Allergens in Antarctic Krill and Mantis Shrimp and Their Amino Acid Sequence Characteristics

Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.

Porcine adipose triglyceride lipase complementary deoxyribonucleic acid clone, expression pattern, and regulation by resveratrol1

Adipose triglyceride lipase (ATGL) was recently identified and described as a major novel triglyceride lipase in animals. In this study, we aimed to study the tissue-specific and developmental expression pattern of porcine ATGL (pATGL) and the effect of resveratrol (RES) on expression of pATGL in vitro. The full-length cDNA sequence of pATGL was 1,958 bp (accession no. EF583921), with a 1,458-bp open reading frame encoding a 486-AA protein (the predicted molecular mass of 53.2 kDa, accession no. ABS58651). Comparison of the deduced AA sequence with the bovine, mouse, rat, dog, and human adipose triglyceride lipase showed 87, 84, 83, 81, and 80% similarity, respectively. Furthermore, the pATGL was highly expressed in porcine adipose tissue, to a lesser degree in kidney, heart, and muscle, and least but detectable in brain. In s.c. adipose tissue, pATGL mRNA was low at birth (1 kg of BW) and then increased, reaching a maximal value at 20 kg of BW (approximately 8 wk old; P < 0.01). In peritoneal and omental adipose tissue, the greatest expression of pATGL was observed at 40 kg of BW (approximately 12 wk old). In vitro, exposure of cultured adipocytes to 40 and 80 muM RES for 24 h increased the mRNA levels of pATGL by 95.3% (P < 0.05) and 146.8% (P < 0.01), respectively. Accordingly, lipid accumulation was decreased by 25.7% (P < 0.05) and 60.8% (P < 0.01), respectively. When treated with RES for 48 h, the mRNA levels of pATGL were increased by 104.1% (P < 0.05) and 163.1% (P < 0.01), respectively. As expected, lipid accumulation was decreased by 9.7% (P > 0.05) and 29.0% (P < 0.05), respectively. These results add to our understanding of the role of pATGL in adipose tissue development and as a potential target for regulating fat deposition and meat quality.

Transcriptional regulation of two cellobiohydrolase encoding genes (cel1 and cel2) from the wood-degrading basidiomycete Polyporus arcularius

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

First hsp70 from two hydrothermal vent shrimps, Mirocaris fortunata and Rimicaris exoculata: Characterization and sequence analysis

The vent shrimps, Mirocaris fortunata and Rimicaris exoculata, live in a highly fluctuating thermal environment and undergo frequent temperature bursts. As a first step in the investigation of the response to heat stress, this work aimed to characterize stress proteins in these two species. Complementary deoxyribonucleic acid (cDNA) clones encoding a 70-kDa heat shock protein (HSP) were isolated and characterized from M. fortunata and R. exoculata. The cDNA clones were of 2055 and 1941 base pairs in length, and contained a 2018-bp complete open reading frame (ORF) and a 1785-bp partial coding sequence, respectively. The amino acid sequences corresponding to these ORF are 645 residues in length for M. fortunata and 595 for R. exoculata, and were clearly characterized as members of the HSP70 family. The C-terminal extremity would identify R. exoculata sequence as a cytoplasm HSP70. The relationships between the crustacean HSP70 sequences were examined by two phylogenetic methods, i.e. Maximum Likelihood and Bayesian methods. The resulting trees suggested that M. fortunata sequence may correspond to constitutively expressed HSP70, named HSC70, whereas R. exoculata sequence may correspond to an inducible form of HSP70. The HSP70 sequences from the hydrothermal shrimps proved to be very similar to the other homologous shrimp sequences, except for the presence of an insertion of unknown function in the ATPase domain of R. exoculata sequence.

Bone Morphogenetic Protein-15 in the Zebrafish Ovary: Complementary Deoxyribonucleic Acid Cloning, Genomic Organization, Tissue Distribution, and Role in Oocyte Maturation

Bone morphogenetic protein-15 (BMP-15) is a member of the TGFbeta family known to regulate ovarian functions in mammals. The structure and function of BMP-15 in lower vertebrates are less known. In this study, we cloned the zebrafish BMP-15 (zfBMP-15) cDNA and depicted its genomic organization. The zfBMP-15 cDNA encodes a protein of 384 amino acids. The mature protein has 46-51% sequence identities to fugu, chicken, and mammalian BMP-15. It also shares 38-46% homology with growth and differentiation factor-9 in fishes, chicken, and mammals. Phylogenetic analysis further confirms that the zfBMP-15 is most closely related to BMP-15 from other species, whereas the growth and differentiation factor-9 peptides from fish to mammals form a distinct branch. Comparison of zfBMP-15 cDNA with zebrafish genome database revealed that zfBMP-15 is encoded by a gene with two exons and one intron, located on chromosome 6. BMP-15 mRNA is expressed in the ovary and testis and, to a lesser extent, brain, liver, gut, heart, and muscle. Real-time PCR revealed that BMP-15 is expressed in follicles at all stages of development with no significant changes over the course of folliculogenesis. Using in situ hybridization and immunocytochemistry, we detected BMP-15 in both oocytes and follicular cells. Incubation of follicles with antiserum against zfBMP15 increased oocyte maturation, whereas incubation with recombinant human BMP-15 suppressed human chorionic gonadotropin-induced oocyte maturation. These findings suggest that BMP-15 plays a role in regulating gonadal functions in fish, in particular oocyte maturation.

Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells

Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.

Molecular cloning and expression of two HSP70 genes in the prawn, Macrobrachium rosenbergii

Two complementary deoxyribonucleic acid (cDNA) clones encoding 2 different 70-kDa heat shock proteins (HSPs) were isolated from the prawn Macrobrachium rosenbergii. The cDNA clones were 2448 and 2173 bp in length and contained 1950- and 1734-bp open reading frames (ORFs), respectively. The ORFs encoded 649- and 577-amino acid polypeptides, which were named Mar-HSC70 and Mar-HSP70, respectively, according to the sequence identities with other known HSC70s and HSP70s and based on their inducibility in response to heat shock stress (at 35 degrees C). Genomic DNA sequence analysis revealed no introns in either gene. The major structural differences between the 2 proteins were a 60-amino acid segment and a 14-amino acid segment present in the N-terminal and C-terminal, respectively, of Mar-HSC70 that were not found in Mar-HSP70. Northern blotting and semiquantitative reverse transcription-polymerase chain reaction analyses indicated that the Mar-HSP70 gene was expressed under heat shock (35 degrees C) stress in a non-tissue-specific manner. In contrast, Mar-HSC70 messenger ribonucleic acid was constitutively expressed in every tissue except muscle, and its expression in response to heat shock (at 35 degrees C) changed only in muscle.

Peptide purification, complementary deoxyribonucleic acid (DNA) and genomic DNA cloning, and functional characterization of ghrelin in rainbow trout.

We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V13R14Q15) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.

Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor.

Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis. Recently it has been indicated that estrogen, estradiol-17beta, is involved in regulating the renewal of spermatogonial stem cells in eel. To determine the genes that directly regulate this process, we used expression screening to identify genes whose expression is regulated by estradiol-17beta in testes. We detected a previously unidentified cDNA clone that is up-regulated by estradiol-17beta stimulation and named it eel spermatogenesis-related substances 34 (eSRS34) cDNA. Homology searching showed that eSRS34 shares amino acid sequence similarity with human platelet-derived endothelial cell growth factor. We examined the function of eSRS34 using several in vitro systems. Recombinant eSRS34 produced by a baculovirus system induced spermatogonial mitosis in testicular organ culture. Furthermore, the addition of an antibody specific for eSRS34 prevented spermatogonial mitosis induced by estradiol-17beta stimulation in a germ cell/somatic cell coculture system. We therefore conclude that eSRS34 is a "spermatogonial stem cell renewal factor."

Studies characterizing 60 kda autoantibodies in subjects with schizophrenia

Background Studies suggest that schizophrenic patients have an increased prevalence of serum antibodies to neuroblastoma cell proteins migrating at 60 kilodaltons (kDa). We present work identifying and characterizing 60 kDa antigen–antibody interactions. Methods Sera from schizophrenic subjects and normal volunteers were screened by Western blotting. Proteins migrating at 60 kDa were characterized by two-dimensional gel electrophoresis and indirect immunofluorescent staining of human epithelial cell (HEp-2) slides. Human brain and bladder cell complementary deoxyribonucleic acid libraries were screened with immunoaffinity-purified antibodies. Complementary deoxyribonucleic acid clones were sequenced and compared with published databases. Proteins were generated by in vitro transcription/translation and expression in an Escherichia coli BL21 system. Immunoprecipitation and immunohistochemistry studies were performed. Results Fifteen percent (17/117) of schizophrenic subjects and 3% (2/62) of normal volunteers had autoantibodies that reacted with 60 kDa proteins [χ 2(1) = 4.4, p = .037]. Five percent of subjects had autoantibodies directed against 60 kDa heat shock protein (HSP60) [χ 2(1) = 3.3, p = .100). Two-dimensional gel electrophoresis identified 13 different proteins migrating at 60 kDa; 5 were splice variants of HSP60, and 2 corresponded with a protein associated with MYC (PAM). Conclusions There is an increased prevalence of autoantibodies that bind to proteins migrating at 60 kDa in subjects with schizophrenia. Potential target antigens include HSP60 and PAM.

Isolation and characterization of Leishmania infantum cDNA encoding a protein homologous to eukaryotic elongation factor 1γ

This paper reports the isolation and characterization of a complementary deoxyribonucleic acid clone showing sequence homology with genes coding for the eukaryotic elongation factor 1γ (EF-1γ). The clone encodes an open reading frame of 404 amino acids corresponding to a deduced molecular mass of 46·2 kDa. Database searches revealed 30–64% sequence identity between the Leishmania infantum EF-1γ and several eukaryotic homologues. Southern blot analysis indicated that 2 genes tandemly organized were present in the L. infantum genome. The 3′ untranslated regions of these 2 genes differed in size. Southern hybridization and pulsed field gel electrophoresis showed that EF-1γ genes are highly conserved among members of the Leishmania genus and must be clustered in a single chromosomal locus.

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI).

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.

Mapping of 12 bovine ribosomal protein genes using a bovine radiation hybrid panel.

Twelve bovine ribosomal protein genes, for which sequence data had been acquired from complementary deoxyribonucleic acid (cDNA) clones isolated from a cattle skin cDNA library, were mapped. As ribosomal protein genes are a group of highly conserved house keeping genes, specific primers were designed to span the intron-exon splice sites and to amplify intronic sequences, in order to obtain bovine-specific polymerase chain reaction (PCR) products. Two of 12 ribosomal protein genes were genotyped in this way and the remaining 10 were mapped using additional primers designed from within the intron. Eleven previously unmapped ribosomal protein genes were localized and one previously reported ribosomal protein gene localization was confirmed. The 12 ribosomal protein genes mapped in this study are spread over 10 chromosomes, including the X chromosome. The locations show conservation of comparative map position in cattle and human.

Cloning of a Novel Growth Hormone-Regulated Rat Complementary Deoxyribonucleic Acid with Homology to the Human 1B-Glycoprotein, Characterizing a New Protein Family

Cloning of a Novel Growth Hormone-Regulated Rat Complementary Deoxyribonucleic Acid with Homology to the Human 1B-Glycoprotein, Characterizing a New Protein Family