Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes

Ford, J. E.; McHeyzer‐Williams, Michael G.; Lieber, Michael R.

doi:10.1016/0378-1119(94)90275-5

Cited by 54 publications

(34 citation statements)

References 23 publications

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“…Notwithstanding the obvious power and utility of PCR, fundamental uncertainties and errors associated with PCR (7,17,19,20,33,36,39,40,42) have significant and mainly negative implications for analysis of, and interpretation of data from, in situ microbial communities and environmental samples. Known biases and inhibitors are likely compounded with additional enzymatic manipulations, such as reverse transcription of RNA into cDNA prior to PCR amplification.…”

Section: Discussionmentioning

confidence: 99%

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

Small¹,

Call

Brockman

et al. 2001

Appl Environ Microbiol

268

184

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We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCRamplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 g of total RNA, representing approximately 7.5 ؋ 10 6

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Section: Discussionmentioning

confidence: 99%

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

Small¹,

Call

Brockman

et al. 2001

Appl Environ Microbiol

268

184

View full text Add to dashboard Cite

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“…Differences at this border were not considered as mutations. Only one A2 sequence, indicative of artifactual crossover (8), was found and was excluded from the data base. We were left with almost 550 mutations from 47 clones, covering a region of 651 nt.…”

Section: Resultsmentioning

confidence: 99%

Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.

González‐Fernández

Gupta²,

Pannell³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The rate and nature of hypermutation of immunoglobulin genes are of prime importance in the affinity maturation of antibodies. Although a considerable body of information has been gathered for K light chains, there is much less data for A chains. We have derived a large data base of somatic mutants of mouse Al light chains from Peyer's patches germinal center B cells. The endogenous Al genes mutate at a rate comparable to that previously found for a K transgene (V,coxl). There are intrinsic hot spots of mutation common to both in-frame and out-of-frame rearrangements; these hot spots cluster in hypermutating domains. In contrast to the pattern seen for VKOxl, the hot spot clusters are found not only in complementarity-determining region (CDR)l but also in CDR2 and CDR3; mutations also cluster in the joining/constant region intron. The differences between the pattern of mutations in VicOxl and Al light chains are discussed.Somatic hypermutation is a key element in antibody diversification and in the maturation of the immune response. Although first demonstrated in A chains (1), somatic mutation has not been as intensively studied as in the heavy-and K light-chain loci (2-4). Indeed, only a small data base of A mutations has been accumulated, and A genes have been suggested to mutate at lower rates than some heavy-chain variable region (V) and K light-chain V genes (2). The A locus is the smallest of the mouse immunoglobulin loci, and germline sequence differences together with junctional imprecision give relatively little diversity. We have recently shown that sequence analysis of V genes cloned from Peyer's patches germinal center B cells provides a rapid way to analyze somatic hypermutation (5). The simplicity of the A locus makes it an easy target for analysis with this approach, and we present the results of such a study.t MATERIALS AND METHODSIsolation of Peyer's Patches A+ B Cells. Peyer's patches were isolated from pooled CBA x C57BL/6 mice (6-12 mo old). The K+ cells were removed by incubation with biotinylated rat anti-mouse K (187.1) antibody (6) followed by streptavidin-magnetic beads (Dynal, Oslo). Germinal center B cells [CD45R(B220)+, peanut agglutinin (PNA)hi] were then sorted as described (5).DNA Isolation and Amplification. DNA was isolated from sorted cells (105), and the rearranged VA genes were amplified by PCR and cloned into M13 essentially as described for VKOX1 (5), except that the extension was for 3 min at 72°C and that oligonucleotides used were primer 1 (5'-CTCGAAT-TCATGGCCTGGATTTCACTTAT), which primes in the leader region and contains an EcoRI site, and primer 2 (5'-CTCGGATCCTTCAGAGGAAGGTGGAAACA), which primes in constant region (C)Al and contains a BamHI site (7). Individual clones were sequenced with LAMBCKF2 (5'-GTCCAAGAAAAACCAGATCATTTATTCAC) and LAMBDAJ1B (5'-GTGAATAAATGATCTGGTTTTTCT-TGGAC) to prime in the frameworks and LAMBDAJ1 (5'-CTCGGATCCACTCACCTAGGACAGTCAGT) and LAMINFOR (5'-GGAGCAGTCTGAAATGAGACAAAG-CAT) to prime in the joining region (J)/C intron. RESULTSBoth In-Fra...

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“…Allelic polymorphism was not considered in the assessment of somatic mutation, because the Ig variable gene alleles have very few nucleotide substitutions (11) and the IMGT database includes various alleles for alignment (10). Chimeric molecules arising from PCR amplification artifacts (12) were not included in any analyses.…”

Section: Ig Sequence Analysismentioning

confidence: 99%

The Microenvironment of Germ Cell Tumors Harbors a Prominent Antigen-Driven Humoral Response

Willis

Mallozzi

Rodig

et al. 2009

The Journal of Immunology

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Germ cell tumors are a heterogeneous group of neoplasms derived from residual primordial tissue. These tumors are commonly found in the brain, testes, or ovaries, where they are termed germinomas, seminomas, or dysgerminomas, respectively. Like several other tumor types, germ cell tumors often harbor an immune cell infiltrate that can include substantial numbers of B cells. Yet little is known about whether the humoral immune response affects germ cell tumor biology. To gain a deeper understanding of the role B cells play in this tumor family, we characterized the immune cell infiltrate of all three germ cell tumor subtypes and defined the molecular characteristics of the B cell Ag receptor expressed by tumor-associated B cells. Immunohistochemistry revealed a prominent B cell infiltrate in the microenvironment of all tumors examined and clear evidence of extranodal lymphoid follicles with germinal center-like architecture in a subset of specimens. Molecular characterization of the Ig variable region from 320 sequences expressed by germ cell tumor-infiltrating B cells revealed clear evidence of Ag experience, in that the cardinal features of an Ag-driven B cell response were present: significant somatic mutation, isotype switching, and codon insertion/deletion. This characterization also revealed the presence of both B cell clonal expansion and variation, suggesting that local B cell maturation most likely occurs within the tumor microenvironment. In contrast, sequences from control tissues and peripheral blood displayed none of these characteristics. Collectively, these data strongly suggest that an adaptive and specific humoral immune response is occurring within the tumor microenvironment.

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Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes

Cited by 54 publications

References 23 publications

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.

The Microenvironment of Germ Cell Tumors Harbors a Prominent Antigen-Driven Humoral Response

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