bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro

To study the regulatory function of Bombyx mori (B. mori) miRNAs (bmo-miR) on the expression of fibroin light chain gene (BmFib-L), the 3’UTR of BmFib-L mRNA was used as the target for online prediction of miRNAs from miRBase using RNAhybrid Software, and miR-2845 was screened out. First, the expression profiles of miR-2845 and BmFib-L in larvae of the 5th instar were analyzed by Real-time quantitative PCR (RT-qPCR). Then recombinant plasmids (pcDNA3.0-pre-miR-2845 and pGL3.0-BmFib-L) were constructed to use for the expression of miR-2845 and BmFib-L 3’UTR, respectively. Cellular-level functional verification of miR-2845 on BmFib-L was carried out using multiple experimental methods (including dual luciferase reporter vectors, artificially synthesized mimics and inhibitors, and target site mutations). Finally, in vivo functional verification was performed by injecting the recombinant vector in 5th instar larvae. BmFib-L expression levels were detected using RT-qPCR in the posterior silk glands (PSG) of the injected larvae. Results showed that the expression of miR-2845 increased between the 1st and 5th day in 5th instar larvae, but began to decline on the 5th day, while the expression of the target gene BmFib-L increased sharply. This suggests that miR-2845 and BmFib-L expression levels show opposing trends, implying a negative regulatory relationship. In BmN cells, miR-2845 significantly down-regulated the expression of BmFib-L; the inhibitory effect of miR-2845 on BmFib-L was disappeared after mutation of the targeting site on 3’UTR of BmFib-L; in individuals, miR-2845 significantly down-regulated BmFib-L expression levels. Our results provide new experimental data for clarifying the molecular regulation mechanism of silk protein expression.

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Intrinsic bent DNA colocalizes with the sequence involved in the Nd-sD mutation in the Bombyx mori fibroin light chain gene

BMB Reports ◽

10.5483/bmbrep.2008.41.5.394 ◽

2008 ◽

Vol 41 (5) ◽

pp. 394-399 ◽

Cited By ~ 3

Author(s):

Joice Felipes Barbosa ◽

Juliana Pereira Bravo ◽

Karen Izumi Takeda ◽

Daniela Bertolini Zanatta ◽

Jose Luis Da Conceicao Silva ◽

...

Keyword(s):

Bombyx Mori ◽

Light Chain ◽

Chain Gene ◽

Bent Dna ◽

Light Chain Gene ◽

Fibroin Light Chain

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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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Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4970-4976.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4970-4976

Author(s):

J Y Zhang ◽

W Bargmann ◽

H R Bose

Keyword(s):

Cell Lines ◽

Light Chain ◽

Lymphoid Cells ◽

Chain Gene ◽

Gene Rearrangements ◽

B Cell Differentiation ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

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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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Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4970 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4970-4976 ◽

Cited By ~ 7

Author(s):

J Y Zhang ◽

W Bargmann ◽

H R Bose

Keyword(s):

Cell Lines ◽

Light Chain ◽

Lymphoid Cells ◽

Chain Gene ◽

Gene Rearrangements ◽

B Cell Differentiation ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

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