A new SEMA7A variant found in Native Americans with alloantibody
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John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status.Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies.Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7AArg347 and variant Sema7ALeu347 proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7AArg347 protein and not the variant one.A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein.[Objective] To investigate the mutations of WD gene in exon 18 and exon 14, and provide information for gene diagnoses. [Methods] Use PCR-SSCP technique to find the abnormity, the abnormity was sequenced. [Results] One case was found abnormal band in exon 18 of 45 patients and 20 normal controls, but no gene mutation was found. [Conclusions] The results suggest that exon 18 and exon 14 may not be frequent mutation points in Chinese Wilson disease.
Exon trapping
Single-strand conformation polymorphism
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To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.
Sf9
Baculoviridae
FLAG-tag
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Objective To construct the prokaryotic expression vector for brain-derived neurotrophic factor(BDNF)gene and express it in E.coli,so as to obtain the recombinant BDNF protein with high yield,low cost,high purity and high biological activity.Methods The cDNA fragment from maturation region of BDNF was obtained by standard PCR method with a full-length human BDNF cDNA as template,and was inserted into plasmid pET-30a(+)by means of gene re-arrangement.The recombinant plasmid pET-BDNF was identified by restriction endonuclease analysis and DNA sequencing.The cloned pET-BDNF plasmid was transformed into E.coli host strain,BL21(DE3)-LysS.Following induction by IPTG,the recombinant protein was expressed and then purified by Ni-NAT affinity chromatography under denaturing conditions.The interest protein was viewed by SDS-PAGE,further characterized by western blot.The proliferation of PC12 cells was evaluated by MTT assay.Results The human BDNF cDNA was amplified and cloned into prokaryotic expression vector.The restriction endonuclease analysis and DNA sequencing proved that the plasmid pET-BDNF was obtained.The recombinant protein was expressed in E.coli system.After Coomassie brilliant blue staining,the recombinant protein showed an exclusive band;western blot analysis with anti-BDNF antibody and anti-his antibody supported that the expressed protein was recombinant BDNF.The recombinant human BDNF protein could enhance the proliferation of PC12 cells.Conclusion The prokaryotic expression vector for human BDNF is successfully constructed.The recombinant human BDNF protein can be expressed and purified in E.coli.and has a good biological activity.
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Tau protein
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We cloned a novel alternative first exon for nitric oxide synthase 1 (NOS1) that is specific for kidney and two novel alternative second exons which can be inserted between the kidney-specific first exon and the exon currently numbered exon 2. The novel exons were localized within 17 kb upstream of exon 2, and their flanking regions and the boundaries of exon 2 were sequenced. NOS1 mRNAs starting with four additional alternative first exons were characterized with respect to tissue distribution and alternative splicing. Altogether, at least 11 different splice variants were found. Those present in kidney were mainly lacking exon 2.
Exon trapping
Exon shuffling
Exon skipping
NOS1
splice
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An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.
Southern blot
Northern blot
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The 5′ untranslated region (UTR) of the glucocorticoid receptor (GR) plays a key role in determining tissue-specific expression and protein isoforms. Analysis of the 5′ UTR of the human GR (hGR) has revealed 11 splice variants of the hGR exon 1, based on seven exon 1s, four of which (1-D to 1-F and 1-H) were previously unknown. All of the exon 1 variants have unique splice donor sites and share a common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the predicted translation from all splice variants is identical. The four new exon 1s show remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17 and 36 bp upstream of the corresponding ends of the rat exon 1 4 . Exon 1-E is only 6 bp longer than its homologue exon 1 5 . Exon 1-F contains two short inserts of 11 and 6 bp when compared with the rat 1 7 . 1-H is 18 bp longer than the corresponding rat 1 11 . In addition to these new exons, we found that the human exon 1-C occurs as three distinct splice variants, covering the region homologous to the rat exons 1 9 and 1 10 . All of the alternative hGR exons 1s presented here were found to be transcribed in human tissue. The human hippocampus expresses mRNA of all the exon 1 variants, while the expression of the other exon 1s seems to be tissue specific. While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5′ region of the hGR is more complex than previously thought, and we suggest that each of these untranslated first exons have a distinct proximal promoter region, providing additional depth to the mechanisms available for tissue-specific expression of the hGR isoforms.
Exon trapping
Exon shuffling
splice
Splice site mutation
Stop codon
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Exon trapping
Exon shuffling
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To examine the alteration and significance of the DPC4 gene in paraffin-embedded tissues of pancreatic carcinomas.Polymerase chain reaction and single-strand conformation polymorphism analysis were used to search for deletions and mutations in the DPC4 gene in 46 cases of pancreatic carcinomas.Thirteen of forty-six (28.3%) cases were found to have homozygous deletions in exon 1, 2, 3, 4, 8 and 11. One was in exon 11, one in exon 1 and 11, one in exon 2 and 3, one in exon 3 and 8, one in exon 1, 2 and 8, one in exon 2, 4 and 11, one in exon 3, 4 and 11, three in exon 3, 4 and 8, one in exon 2, 3, 4, and 8, one in exon 2, 3, 8 and 11, one in exon 2, 3, 4, 8 and 11. Intragenic mutations were found in 10 of 46 cases (21.7%). One case was in exon 1, one in exon 2, three in exon 8, four in exon 11, and one in exon 4 and 11. The total frequency of intragenic changes of DPC4 in paraffin-embedded tissues was 45.6% (21/46).Inactivation of tumor-suppressor gene DPC4 may play an important role during the tumorigenesis of pancreatic carcinomas.
Exon trapping
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Exon trapping
Exon shuffling
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