Inverse PCR amplification of low-abundancy message of γδ T cell receptor genes

Weber-Arden, J.; Wilbert, O.M.; Kabelitz, Dieter; Arden, Bernhard

doi:10.1016/0022-1759(96)00151-2

Cited by 6 publications

(3 citation statements)

References 10 publications

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“…Isolation of Bacaq α‐amylase gene ( baqA ) was conducted through several PCR steps using degenerate primers (degPCR) and inverse PCR (invPCR) (Weber‐Arden et al . ). The nucleotide sequences of degenerate primers were 5′‐GAYTTYRTYGTSAATCAYGTYGG‐3′ (degFA), 5′‐GATGGRTAYCGTCTRGATACYG‐3′ (degFB), 5′‐AATCGWMYCATATCATGGTTATC‐3′ (degR2) and 5′‐CAGATCCRTAGTAAASAATSGG‐3′ (degR4).…”

Section: Methodsmentioning

confidence: 97%

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

et al. 2012

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Aims: The aims were to isolate a raw starch-degrading a-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. Methods and Results:A 1539 complete open reading frame of a starchdegrading a-amylase gene baqA from B. aquimaris MKSC 6Á2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 a-amylase (BaqA) has no starchbinding domain, and together with a few putative a-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. Conclusions: A novel raw starch-degrading B. aquimaris MKSC 6.2 a-amylase BaqA is proposed to be a member of new GH13 subfamily. Significance and Impact of the Study: This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.

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Section: Methodsmentioning

confidence: 97%

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

et al. 2012

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“…The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive ␥␦ precursor waves bearing this canonical DV101 rearrangement will coincide on day 16 T he ␥␦ T cells can be detected in the thymus around day 13 of gestation, 3 days before ␣␤ T cells are found. During fetal ontogeny successive waves of ␥␦ cells arise bearing TCR composed of different variable (V␥ and V␦) regions (reviewed in Refs.…”

Section: V␦ Repertoire During Thymic Ontogeny Suggests Three Novel Wamentioning

confidence: 94%

“…Outwardly oriented C␦-specific primers (a 5Ј antisense and a 3Ј sense primer) were used to amplify around the circle from the C␦ gene segment into the unknown flanking gene segments. The protocol of iPCR has been established for investigating TCR-␣␤ junctional diversity in human peripheral blood (15) and has been improved at certain steps to make up for the minimal amount of ␥␦ message present in fetal murine thymus (16).…”

Section: Inverse Pcrmentioning

confidence: 99%

Vδ Repertoire During Thymic Ontogeny Suggests Three Novel Waves of γδ TCR Expression

Weber-Arden

Wilbert

Kabelitz

et al. 2000

The Journal of Immunology

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Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR δ-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive γδ precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal δ-chain transcripts used a second wave of diverse Vα gene segments that are exclusively located in the δ locus-proximal gene cluster of intermingled single members of different Vα subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the α locus, but their usage does not show the position-dependent bias of the other Vα-to-δ rearrangements.

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Complementary DNA sequences encoding rat T-cell receptor δ-chain D1 -, D2 -, J1 -, and C -region proteins

2000

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Inverse PCR amplification of low-abundancy message of γδ T cell receptor genes

Cited by 6 publications

References 10 publications

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

Vδ Repertoire During Thymic Ontogeny Suggests Three Novel Waves of γδ TCR Expression

Complementary DNA sequences encoding rat T-cell receptor δ-chain D1 -, D2 -, J1 -, and C -region proteins

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