A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible.

Objective.Transient B cell depletion by rituximab (RTX) has become a specific treatment of rheumatoid arthritis (RA). Although phenotypic repopulation kinetics of B cell subsets are well documented, precise molecular analyses of the reconstituting immunoglobulin (Ig) genes encoding the B cell receptor in RA are sparse.Methods.A total of 708 individual CD19+CD27+ (memory) and CD19+CD27– (naive) B cells from 2 patients with RA were analyzed at baseline and 7 months after RTX at B cell repopulation. Ig light chain variable kappa (Vκ) and lambda (Vλ) light chain gene rearrangements were amplified, sequenced, and analyzed with a focus on receptor revision.Results.The naive as well as the memory repertoire repopulated polyclonally with diverse use of variable light chain gene families and minigenes. During the reconstitution phase, B cells used significantly fewer Jκ distal Vκ genes (p = 0.0006), with a higher frequency of somatic hypermutation of rearrangements employing Jκ5 compared to baseline in memory B cells. The use of Vλ rearrangements in regenerating B cells was also biased toward use of Vλ genes of the proximal cassette. In general, reemerging CD27+ Ig light chain genes were substantially more highly mutated than before RTX therapy (p < 0.0001, baseline vs during reconstitution).Conclusion.Our data indicate that RTX therapy leads to generation of distinct Vκ/Jκ and Vλ/Jλ gene repertoires consistent with replenishment of antigen-experienced B cells by germinal centers. At baseline, the imprints of receptor revision appeared to be more striking, which indicates that receptor revision is active in patients with RA and can be reduced by RTX.

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Faculty Opinions recommendation of Bruton's tyrosine kinase and SLP-65 regulate pre-B cell differentiation and the induction of Ig light chain gene rearrangement.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014431.370884 ◽

2006 ◽

Author(s):

Ranjan Sen

Keyword(s):

Cell Differentiation ◽

Tyrosine Kinase ◽

B Cell ◽

Light Chain ◽

Gene Rearrangement ◽

Chain Gene ◽

B Cell Differentiation ◽

Bruton's Tyrosine Kinase ◽

Light Chain Gene ◽

Chain Gene Rearrangement

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Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3168-3182.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3168-3182

Author(s):

E E Strehler ◽

M Periasamy ◽

M A Strehler-Page ◽

B Nadal-Ginard

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Mammalian Species ◽

In Vitro Transcription ◽

Chain Gene ◽

Promoter Regions ◽

Light Chain Gene ◽

Direct Initiation

DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA.

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Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2513-2525.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2513-2525

Author(s):

T Braun ◽

E Tannich ◽

G Buschhausen-Denker ◽

H H Arnold

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Mutational Analysis ◽

Nuclear Proteins ◽

Chain Gene ◽

Cardiac Myosin ◽

Mobility Shift ◽

Light Chain Gene ◽

Sequence Elements

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

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Ongoing diversification of the rearranged immunoglobulin light-chain gene in a bursal lymphoma cell line

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3224-3231.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3224-3231

Author(s):

S Kim ◽

E H Humphries ◽

L Tjoelker ◽

L Carlson ◽

C B Thompson

Keyword(s):

B Cell ◽

Light Chain ◽

Gene Segment ◽

B Cell Development ◽

Bursa Of Fabricius ◽

Chain Gene ◽

Lymphoma Cell Line ◽

Immunoglobulin Light Chain ◽

Light Chain Gene

The chicken immunoglobulin light-chain gene (IgL) encodes only a single variable gene segment capable of recombination. To generate an immune repertoire, chickens diversify this unique rearranged VL gene segment during B-cell development in the bursa of Fabricius. Sequence analysis of IgL cDNAs suggests that both gene conversion events derived from VL segment pseudogene templates (psi VL) and non-template-derived single-base-pair substitutions contribute to this diversity. To facilitate the study of postrecombinational mechanisms of immunoglobulin gene diversification, avian B-cell lines were examined for the ability to diversify their rearranged IgL gene during in vitro passage. One line that retains this ability, the avian leukosis virus-induced bursal lymphoma cell line DT40, has been identified. After passage for 1 year in culture, 39 of 51 randomly sequenced rearranged V-J segments from a DT40 population defined novel subclones of the parental tumor. All cloned V-J segments displayed the same V-J joint, confirming that the observed diversity arose after V-J rearrangement. Most sequence variations that we observed (203 of 220 base pairs) appeared to result from psi VL-derived gene conversion events; 16 of the 17 novel single nucleotide substitutions were transitions. Based on these data, it appears that immunoglobulin diversification during in vitro passage of DT40 cells is representative of the diversification that occurs during normal B-cell development in the bursa of Fabricius.

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Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.902 ◽

1993 ◽

Vol 13 (2) ◽

pp. 902-910 ◽

Cited By ~ 42

Author(s):

A M Rushforth ◽

B Saari ◽

P Anderson

Keyword(s):

Polymerase Chain Reaction ◽

Caenorhabditis Elegans ◽

Light Chain ◽

Myosin Light Chain ◽

Null Alleles ◽

Chain Gene ◽

Splice Sites ◽

Chain Reaction ◽

Light Chain Gene ◽

Polymerase Chain

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.

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