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1.
Mol Immunol ; 50(4): 185-92, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22336572

ABSTRACT

We have previously reported accelerated acquisition of new autoreactivity upon immunization with 4-hydroxy-2-nonenal (HNE)-modified Ro60, as well as differential induction of lupus or Sjögren's syndrome by immunization with Ro60 containing varying amounts of HNE. Since the number of HNE molecules on Ro60 appears to be important, we hypothesized that specific sequences on Ro60 are targets for HNE-modification. Using surface plasmon resonance (SPR) we have also shown intramolecular protein-protein interaction between Ro60 and Ro multiple antigenic peptides (MAPs). We also hypothesized that intramolecular protein-protein interaction would be abolished by HNE-modification. To test this hypotheses we investigated (a) the epitopes of Ro60, using 19 Ro MAPs in an in vitro assay (involving HNE-modification of MAPs following immobilization on ELISA plates) to identify targets of HNE modification on Ro60 and (b) the protein-protein interaction between unmodified Ro60 MAPs, immobilized on the sensor surface of BIAcore, and unmodified Ro60 or HNE-modified Ro60 using SPR. New data obtained with SPR strengthens our earlier observation that immunization with HNE-Ro60 induces a stronger response. Unmodified Ro60 bound to several Ro60 MAPs through protein-protein interaction analyzed using SPR. This interaction was totally abrogated using HNE-modified Ro60 suggesting that sequences on Ro had become modified with HNE. When 19 Ro60 MAPs were modified in vitro with HNE, it was found that 10/19 MAPs significantly bound HNE covalently (p<0.001 compared to MAPs binding HNE poorly). The amino acid sequences 126-137, 166-272 and 401-495 on Ro60 were strongly HNE modified. Using computational model system based on the recently published crystal structure for Ro60 enabled us to identify regions on the Ro60 molecule represented by the HNE-modified Ro MAPs, which are part of the exposed tertiary structure of the Ro60 protein.


Subject(s)
Aldehydes/chemistry , Aldehydes/immunology , Autoantigens/chemistry , Autoantigens/immunology , Ribonucleoproteins/chemistry , Ribonucleoproteins/immunology , Amino Acid Sequence , Animals , Autoantibodies/blood , Autoantibodies/immunology , Cattle , Cross-Linking Reagents/chemistry , Epitopes, B-Lymphocyte/immunology , Lupus Erythematosus, Systemic/immunology , Molecular Sequence Data , Protein Structure, Secondary , Rabbits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Surface Plasmon Resonance
2.
PLoS One ; 6(3): e18003, 2011 Mar 28.
Article in English | MEDLINE | ID: mdl-21464925

ABSTRACT

INTRODUCTION: Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögren's syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved. METHODS: Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts. RESULTS: Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity. CONCLUSIONS: Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.


Subject(s)
Immunization , Interferon-gamma/immunology , Ribonucleoproteins/immunology , Salivary Glands/immunology , Salivary Glands/physiopathology , Sjogren's Syndrome/immunology , Sjogren's Syndrome/physiopathology , Adjuvants, Immunologic , Animals , Autoantibodies/immunology , Disease Models, Animal , Female , Injections , Interferon-gamma/biosynthesis , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Salivation , Spleen/immunology , Spleen/pathology , Th1 Cells/immunology
3.
Acta Paediatr ; 100(6): 819-23, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21375582

ABSTRACT

AIM: To determine the rate of Klinefelter's syndrome among men with systemic lupus erythematosus (SLE), and to determine whether the manifestations of SLE in these men are different from that seen in 46,XY men. METHODS: A total of 276 men with SLE underwent a real-time PCR procedure to screen for more than one X chromosome. Those with results consistent with two X chromosomes were further characterized by karyotype and FISH. Clinical manifestations of SLE were determined by interview, questionnaire and medical chart review. Each man with Klinefelter's and SLE was matched to four 46,XY men with SLE. Rates of SLE manifestations were compared with chi-square analyses. RESULTS: We found seven of the 286 men with SLE had Klinefelter's syndrome. Four of these seven were nonmosaic 47,XXY, while two were mosaic 46,XY/47,XXY and one was 46,XX/47,XXY. The men with 47,XXY did not have severe manifestations of SLE including no proliferative renal disease, neurological disease, thrombocytopenia, autoimmune haemolytic anaemia, discoid skin disease or anti-RNP/Sm. CONCLUSION: 47,XXY is found in excess among men with SLE. Men commonly have SLE that is more severe than that found among women, but the 47,XXY men had less severe SLE than other men.


Subject(s)
Klinefelter Syndrome/epidemiology , Lupus Erythematosus, Systemic/epidemiology , Humans , Klinefelter Syndrome/complications , Klinefelter Syndrome/genetics , Lupus Erythematosus, Systemic/complications , Male , Mosaicism , Severity of Illness Index
4.
Free Radic Biol Med ; 50(10): 1222-33, 2011 May 15.
Article in English | MEDLINE | ID: mdl-20946951

ABSTRACT

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.


Subject(s)
Aldehydes/immunology , Aldehydes/metabolism , Lipid Peroxidation , Lupus Erythematosus, Systemic/immunology , Ribonucleoproteins/immunology , Ribonucleoproteins/metabolism , Sjogren's Syndrome/immunology , Aldehydes/pharmacology , Animals , Autoimmunity/drug effects , Autoimmunity/immunology , Female , Lupus Erythematosus, Systemic/physiopathology , Mice , Mice, Inbred BALB C , Salivary Glands/drug effects , Salivary Glands/immunology , Salivary Glands/physiopathology , Sjogren's Syndrome/physiopathology
6.
J Rheumatol ; 37(7): 1480-7, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20472921

ABSTRACT

OBJECTIVE: Systemic lupus erythematosus (SLE) is more common among women than men, a ratio of about 10 to 1. We undertook this study to describe familial male SLE within a large familial SLE cohort. METHODS: SLE families (2 or more patients) were identified from the Lupus Multiplex Registry and Repository. Genomic DNA and blood samples were obtained using standard methods. Autoantibodies were determined by multiple methods. Medical records were abstracted for SLE clinical data. Fluorescent in situ hybridization (FISH) was performed with X and Y centromere-specific probes, and a probe specific for the Toll-like receptor 7 gene on the X chromosome. RESULTS: Among 523 SLE families, we found 5 families in which all the SLE patients were male. FISH found no yaa gene equivalent in these families. SLE-unaffected primary female relatives from the 5 families with only-male SLE patients had a statistically increased rate of positive antinuclear antibodies compared to SLE-unaffected female relatives in other families. White men with SLE were 5 times more likely to have an offspring with SLE than White women with SLE, but there was no difference in this likelihood among Black men. CONCLUSION: Because women in the all-male families had positive antinuclear antibodies, and men are more likely to have children with SLE, these data suggest genetic susceptibility factors that act only in men.


Subject(s)
Lupus Erythematosus, Systemic/genetics , Black or African American/genetics , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Autoantibodies/blood , Autoantibodies/genetics , Chromosomes, Human, X , Chromosomes, Human, Y , Cohort Studies , Female , Genetic Predisposition to Disease , Humans , In Situ Hybridization, Fluorescence , Lupus Erythematosus, Systemic/blood , Male , Pedigree , Registries , White People/genetics
7.
Mol Nutr Food Res ; 54(8): 1202-9, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20146265

ABSTRACT

Chronic and complex autoimmune diseases, currently treated palliatively with immunosuppressives, require multi-targeted therapy for greater effectiveness. The naturally occurring polyphenol curcumin has emerged as a powerful "nutraceutical" that interacts with multiple targets to regress diseases safely and inexpensively. Up to 8 g/day of curcumin for 18 months was non-toxic to humans. However, curcumin's utility is limited by its aqueous insolubility. We have demonstrated a heat-mediated 12-fold increase in curcumin's aqueous solubility. Here, we show by SDS-PAGE and surface plasmon resonance that heat-solubilized curcumin binds to proteins. Based on this binding we hypothesized that heat-solubilized curcumin or turmeric would prevent autoantibody targeting of cognate autoantigens. Heat-solubilized curcumin/turmeric significantly decreased binding of autoantibodies from Sjögren's syndrome (up to 43/70%, respectively) and systemic lupus erythematosus (up to 52/70%, respectively) patients as well as an animal model of Sjögren's syndrome (up to 50/60%, respectively) to their cognate antigens. However, inhibition was not specific to autoimmunity. Heat-solubilized curcumin/turmeric also inhibited binding of commercial polyclonal anti-spectrin to spectrin (50/56%, respectively). Thus, we suggest that the multifaceted heat-solubilized curcumin can ameliorate autoimmune disorders. In addition, the non-toxic curcumin could serve as a new protein stain in SDS-PAGE even though it is less sensitive than the Coomassie system which involves toxic chemicals.


Subject(s)
Antigen-Antibody Reactions/drug effects , Autoimmune Diseases/immunology , Curcumin/chemistry , Curcumin/pharmacology , Hot Temperature , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Animals , Autoantibodies/immunology , Autoantibodies/metabolism , Autoantigens/immunology , Autoantigens/metabolism , Autoimmune Diseases/diet therapy , Curcuma/chemistry , Curcuma/metabolism , Curcumin/metabolism , Dietary Supplements , Electrophoresis, Polyacrylamide Gel/methods , Humans , Immunologic Factors/metabolism , Indicators and Reagents , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred BALB C , Plant Extracts/chemistry , Plant Extracts/metabolism , Plant Extracts/pharmacology , Sjogren's Syndrome/immunology , Solubility , Spectrin/immunology , Surface Plasmon Resonance
8.
Methods Mol Biol ; 536: 433-40, 2009.
Article in English | MEDLINE | ID: mdl-19378080

ABSTRACT

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.


Subject(s)
Antigens/analysis , Blotting, Western/methods , Coloring Agents/chemistry , Staining and Labeling/methods , Collodion/chemistry , Polyvinyls/chemistry
9.
Biotechnol J ; 4(8): 1210-4, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19370710

ABSTRACT

C1q is of interest in systemic lupus erythematosus (SLE) research due to deficiencies in its activity being associated with the disease. Current published protocols for measuring C1q vary greatly in their results and ease of reproducibility. Due to this, average C1q concentrations have been reported between 56 and 276 microg/mL in non-SLE serum. We present an improved method for quantifying C1q concentrations, which employs a sandwich ELISA. This method has improved precision, cost efficiency, up-scaling, reproducibility, and uses significantly lesser volumes of serum sample when compared to RID and other methods for quantifying C1q. We report an average concentration of 113 +/- 40 microg/mL for C1q in non-SLE serum. The assay designed here will be useful in the high-throughput measurement of serum C1q in SLE cases.


Subject(s)
Complement C1q/biosynthesis , Complement C1q/immunology , Enzyme-Linked Immunosorbent Assay/methods , Lupus Erythematosus, Systemic/diagnosis , Lupus Erythematosus, Systemic/immunology , Animals , Antibodies, Monoclonal/chemistry , Biotechnology/methods , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay/economics , Enzyme-Linked Immunosorbent Assay/instrumentation , Humans , Mice , Reproducibility of Results , Spectrophotometry/methods , Time Factors
10.
BMC Med Genet ; 9: 62, 2008 Jul 07.
Article in English | MEDLINE | ID: mdl-18606005

ABSTRACT

BACKGROUND: Systemic lupus erythematosus (SLE) is a multifactorial disorder characterized by the presence of autoantibodies. We and others have implicated free radical mediated peroxidative damage in the pathogenesis of SLE. Since harmful free radical products are formed during this oxidative process, including 4-hydroxy 2-nonenol (4-HNE) and malondialdehyde (MDA), we hypothesized that specific HNE-protein adducts would be present in SLE red blood cell (RBC) membranes. Catalase is located on chromosome 11p13 where linkage analysis has revealed a marker in the same region of the genome among families with thrombocytopenia, a clinical manifestation associated with severe lupus in SLE affected pedigrees. Moreover, SLE afflicts African-Americans three times more frequently than their European-American counterparts. Hence we investigated the effects of a genetic polymorphism of catalase on risk and severity of SLE in 48 pedigrees with African American ancestry. METHODS: Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis was used to identify the protein modified by HNE, following Coomassie staining to visualize the bands on the acrylamide gels. Genotyping analysis for the C --> T, -262 bp polymorphism in the promoter region of catalase was performed by PCR-RFLP and direct PCR-sequencing. We used a "pedigree disequilibrium test" for the family based association analysis, implemented in the PDT program to analyze the genotyping results. RESULTS: We found two proteins to be HNE-modified, migrating around 80 and 50 kD respectively. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the Coomassie stained 80 kD band revealed that the target of HNE modification was catalase, a protein shown to associate with RBC membrane proteins. All the test statistics carried out on the genotyping analysis for the C --> T, -262 bp polymorphism in the promoter region of catalase were non-significant (p > 0.05) in our data, which suggested that this SNP is not associated with SLE. CONCLUSION: Our results indicate that catalase is one of the proteins modified due to oxidative stress. However, catalase may not be a susceptibility gene for SLE. Nonetheless, catalase is oxidatively modified among SLE patients. This suggests a possible role between oxidative modification of catalase and its affects on enzymatic activity in SLE. An oxidatively modified catalase could be one of the reasons for lower enzymatic activity among SLE subjects, which in turn could favor the accumulation of deleterious hydrogen peroxide. Furthermore, HNE-products are potential neoantigens and could be involved in the pathogenesis of SLE. Decrease in catalase activity could affect the oxidant-antioxidant balance. Chronic disturbance of this balance in patients with SLE may work favorably for the premature onset of atherogenesis with severe vascular effect.


Subject(s)
Aldehydes/blood , Catalase/genetics , Lipid Peroxidation/genetics , Lupus Erythematosus, Systemic/genetics , Catalase/blood , Erythrocyte Membrane/metabolism , Genetic Predisposition to Disease , Humans , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/enzymology , Oxidative Stress , Polymorphism, Single Nucleotide , Risk Factors
11.
Med Hypotheses ; 68(6): 1195-9, 2007.
Article in English | MEDLINE | ID: mdl-17306929

ABSTRACT

Fed up with life on earth, four scientists attempt to make it to space to live in the International Space Station (ISS) and carry out experiments. The difficulties in getting selected by NASA, the rigourous training to fly and the risks of the journey to life and health are the rate limiting steps in their quest. They propose commercialization of space and also ferrying cows to space for food as well as generation of biogas. The anaerobic environment is particularly suitable for biogas generation and if successful they plan to get NASA to launch space vehicles to Mars using this natural fuel with the ISS as the staging area.


Subject(s)
Rest/physiology , Space Flight/methods , Spacecraft/instrumentation , Work/physiology , Animals , Cattle , Female , Humans , Space Flight/education , Space Flight/instrumentation
12.
Am J Med Genet A ; 140(6): 580-5, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16470701

ABSTRACT

Encoded by the peptidase D (PEPD) gene located at 19q12-q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C-terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma-like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR-amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop-codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation.


Subject(s)
Codon, Nonsense , Dipeptidases/genetics , Ethnicity/genetics , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Base Sequence , Child , DNA Mutational Analysis , Dipeptidases/blood , Dipeptidases/deficiency , Family Health , Female , Hepatomegaly/pathology , Humans , Male , Pedigree , Skin Ulcer/pathology , Splenomegaly/pathology
14.
J Biol Chem ; 280(43): 36141-9, 2005 Oct 28.
Article in English | MEDLINE | ID: mdl-16120602

ABSTRACT

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.


Subject(s)
Gene Expression Regulation, Enzymologic , Lysosomes/chemistry , N-Acetylglucosaminyltransferases/chemistry , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Aspartic Acid/chemistry , Blotting, Northern , Catalytic Domain , Cattle , Cell Line , Cloning, Molecular , DNA, Complementary/metabolism , Gene Library , Humans , Lysine/chemistry , Mice , Models, Genetic , Molecular Sequence Data , Oligonucleotides/chemistry , Peptides/chemistry , Plasmids/metabolism , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Transfection
15.
Nucleic Acids Res ; 30(12): e52, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12060690

ABSTRACT

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.


Subject(s)
Caenorhabditis elegans/genetics , DNA Mutational Analysis/methods , DNA, Helminth , Polymerase Chain Reaction/methods , Sequence Deletion , Alleles , Animals , DNA , Genes, Helminth , Sensitivity and Specificity
16.
J Biol Chem ; 277(1): 178-86, 2002 Jan 04.
Article in English | MEDLINE | ID: mdl-11677243

ABSTRACT

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T., Cummings, R. D., and Canfield, W. M. (2002) J. Biol. Chem. 277, 169-177) were used to identify the core 1 beta3-Gal-T sequences in the human expressed sequence tag data bases. A 1794-bp human core 1 beta3-Gal-T cDNA sequence was determined by sequencing the expressed sequence tag and performing 5'-rapid amplification of cDNA ends. The core 1 beta3-Gal-T predicts a 363-amino acid type II transmembrane protein. Expression of both the full-length and epitope-tagged soluble forms of the putative enzyme in human 293T cells generated core 1 beta3-Gal-T activity that transferred galactose from UDP-Gal to GalNAcalpha1-O-phenyl, and a synthetic glycopeptide with Thr-linked GalNAc and the product was shown to have the core 1 structure. Northern analysis demonstrated widespread expression of core 1 beta3-Gal-T in tissues with a predominance in kidney, heart, placenta, and liver. Highly homologous cDNAs were identified and cloned from rat, mouse, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that the enzyme is widely distributed in metazoans. The core 1 beta3-Gal-T sequence has minimal homology with conserved sequences found in previously described beta3-galactosyltransferases, suggesting this enzyme is only distantly related to the known beta3-galactosyltransferase family.


Subject(s)
Galactosyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , Galactosyltransferases/analysis , Galactosyltransferases/chemistry , Glomerulonephritis, IGA/etiology , Humans , Molecular Sequence Data , Plasmids , Rats
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