Transient expression of maize anthocyanin regulatory genes influences anthocyanin production in white clover and peas

1998 ◽  
Vol 25 (3) ◽  
pp. 335 ◽  
Author(s):  
John de Majnik ◽  
G.J. Tanner ◽  
R.G. Joseph ◽  
P.J. Larkin ◽  
J.J. Weinman ◽  
...  
Keyword(s):  
Transient Expression ◽  
White Clover ◽  
Anthocyanin Biosynthesis ◽  
Cell Suspensions ◽  
Regulatory Genes ◽  
Coding Region ◽  
Specific Expression ◽  
Tissue Specific ◽  
Maize Cell ◽  
Anthocyanin Production

Transient expression of the maize anthocyanin regulatory genes, B-Peru and C1, was examined in maize cell suspensions, and in pea and white clover tissues in order to determine if these maize regulators can function in legume tissues. Optimal ratios and amounts of B-Peru and C1 genes for anthocyanin production were determined in maize cell suspensions. Activation of anthocyanin biosynthesis in legume tissues was observed following expression of the B-Peru and C1 genes, delivered in the pre-determined optimal ratio and amounts by microparticle bombardment. Tissue-specific expression of these genes was observed in the legumes tested and the exact sites of anthocyanin expression in pea depended on the cultivar tested. Maize anthocyanin regulatory genes can activate anthocyanin biosynthesis in legume tissues, but appear to be subject to tissue specific controls. Three of the four varieties of pea tested were able to accumulate anthocyanin when the B-Peru coding region alone was biolistically expressed.

scholarly journals Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

1987 ◽  
Vol 7 (7) ◽  
pp. 2425-2434 ◽  
Author(s):  
J M Heard ◽  
P Herbomel ◽  
M O Ott ◽  
A Mottura-Rollier ◽  
M Weiss ◽  
...  
Keyword(s):  
Transient Expression ◽  
Tissue Specificity ◽  
Transcriptional Start Site ◽  
Expression System ◽  
Hepatoma Cells ◽  
Sequence Motif ◽  
Specific Expression ◽  
Tissue Specific ◽  
Albumin Gene ◽  
Ccaat Box

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

scholarly journals A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

2021 ◽  
Author(s):  
Justin M Shaffer ◽  
Iva Greenwald
Keyword(s):  
Gene Expression ◽  
Specific Protein ◽  
Specific Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Nonsense Mediated Decay ◽  
Conditional Gene Expression ◽  
Stop Cassette

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

scholarly journals Duplicated flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase genes in barley genome

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6266 ◽  
Author(s):  
Alexander V. Vikhorev ◽  
Ksenia V. Strygina ◽  
Elena K. Khlestkina
Keyword(s):  
Cytochrome P450 ◽  
Anthocyanin Biosynthesis ◽  
Gene Families ◽  
Gene Copy ◽  
Aleurone Layer ◽  
Specific Expression ◽  
Tissue Specific ◽  
Barley Genome ◽  
Tissue Specific Expression ◽  
Different Parts

Background Anthocyanin compounds playing multiple biological functions can be synthesized in different parts of barley (Hordeum vulgare L.) plant. The diversity of anthocyanin molecules is related with branching the pathway to alternative ways in which dihydroflavonols may be modified either with the help of flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H)—the cytochrome P450-dependent monooxygenases. The F3′H and F3′5′H gene families are among the least studied anthocyanin biosynthesis structural genes in barley. The aim of this study was to identify and characterise duplicated copies of the F3′H and F3′5′H genes in the barley genome. Results Four copies of the F3′5′H gene (on chromosomes 4HL, 6HL, 6HS and 7HS) and two copies of the F3′H gene (on chromosomes 1HL and 6HS) were identified in barley genome. These copies have either one or two introns. Amino acid sequences analysis demonstrated the presence of the flavonoid hydroxylase-featured conserved motifs in all copies of the F3′H and F3′5′H genes with the exception of F3′5′H-3 carrying a loss-of-function mutation in a conservative cytochrome P450 domain. It was shown that the divergence between F3′H and F3′5′H genes occurred 129 million years ago (MYA) before the emergence of monocot and dicot plant species. The F3′H copy approximately occurred 80 MYA; the appearance of F3′5′H copies occurred 8, 36 and 91 MYA. qRT-PCR analysis revealed the tissue-specific activity for some copies of the studied genes. The F3′H-1 gene was transcribed in aleurone layer, lemma and pericarp (with an increased level in the coloured pericarp), whereas the F3′H-2 gene was expressed in stems only. The F3′5′H-1 gene was expressed only in the aleurone layer, and in a coloured aleurone its expression was 30-fold higher. The transcriptional activity of F3′5′H-2 was detected in different tissues with significantly higher level in uncoloured genotype in contrast to coloured ones. The F3′5′H-3 gene expressed neither in stems nor in aleurone layer, lemma and pericarp. The F3′5′H-4 gene copy was weakly expressed in all tissues analysed. Conclusion F3′H and F3′5′H-coding genes involved in anthocyanin synthesis in H. vulgare were identified and characterised, from which the copies designated F3′H-1, F3′H-2, F3′5′H-1 and F3′5′H-2 demonstrated tissue-specific expression patterns. Information on these modulators of the anthocyanin biosynthesis pathway can be used in future for manipulation with synthesis of diverse anthocyanin compounds in different parts of barley plant. Finding both the copies with tissue-specific expression and a copy undergoing pseudogenization demonstrated rapid evolutionary events tightly related with functional specialization of the duplicated members of the cytochrome P450-dependent monooxygenases gene families.

scholarly journals Ratio of Myc and Myb Transcription Factors Regulates Anthocyanin Production in Orchid Flowers

2008 ◽  
Vol 133 (1) ◽  
pp. 133-138 ◽  
Author(s):  
Hongmei Ma ◽  
Margaret Pooler ◽  
Robert Griesbach
Keyword(s):  
Transcription Factors ◽  
Transient Expression ◽  
Internal Control ◽  
Expression System ◽  
Regulatory Gene ◽  
Regulatory Genes ◽  
Anthocyanin Accumulation ◽  
Green Florescent Protein ◽  
Negative Effect ◽  
Anthocyanin Production

Many studies have examined anthocyanin gene expression in colorless tissues by introducing anthocyanin regulatory genes of the MYC/R and MYB/C1 families. Expression of the two regulatory genes under the control of a strong promoter generally results in high anthocyanin accumulation. However, such approaches usually have a negative effect on growth and development of the recovered plants. In this study the author used two promoters of different strengths—a weak (Solanum tuberosum L. polyubiquitin Ubi3) and a strong (double 35S) promoter—and generated two sets of expression constructs with the Zea mays L. anthocyanin regulatory genes MycLc and MybC1 . A transient expression system was developed using biolistic bombardment of white Phalaenopsis amabilis (L.) Blume flowers, which the authors confirmed to be anthocyanin regulatory gene mutants. Transient expression of different combinations of the four constructs would generate three different MycLc -to-MybC1 ratios (>1, 1, <1). The enhanced green florescent protein gene (EGFP) was cotransformed as an internal control with the two anthocyanin regulatory gene constructs. These results demonstrate that the ratio of the two transcription factors had a significant influence on the amount of anthocyanin produced. Anthocyanin accumulation occurred only when MybC1 was under the control of the 35S promoter, regardless of whether MycLC was driven by the 35S or Ubi3 promoter.

scholarly journals Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

1987 ◽  
Vol 7 (11) ◽  
pp. 3916-3922 ◽  
Author(s):  
L J Mullins ◽  
G Veres ◽  
C T Caskey ◽  
V Chapman
Keyword(s):  
Methylation Status ◽  
Coding Region ◽  
Specific Expression ◽  
X Chromosomes ◽  
Partial Methylation ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Ornithine Carbamoyl Transferase ◽  
Inactive X Chromosome ◽  
Inactive Genes

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.

scholarly journals Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles

1989 ◽  
Vol 86 (17) ◽  
pp. 6681-6685 ◽  
Author(s):  
Theodore M. Klein ◽  
Bradley A. Roth ◽  
Michael E. Fromm
Keyword(s):  
Luciferase Activity ◽  
Anthocyanin Biosynthesis ◽  
Regulation Of Gene Expression ◽  
Coding Region ◽  
Chimeric Genes ◽  
Endogenous Genes ◽  
Specific Regulation ◽  
Aleurone Tissue ◽  
Anthocyanin Production ◽  
Anthocyanin Biosynthetic Genes

We have employed microprojectiles to deliver genes involved in anthocyanin biosynthesis to cells within intact aleurone and embryo tissues of maize. Clones of the A1 or Bz1 genes were introduced into aleurone tissue that lacked anthocyanins due to mutations of the endogenous A1 or Bz1 gene. Following bombardment, cells within the aleurone developed purple pigmentation, indicating that the mutation in the a1 or bz1 genotypes was corrected by the introduced gene. To analyze the expression of these genes in different genetic backgrounds, chimeric genes containing the 5′ and 3′ regions of the A1 or Bz1 genes fused to a luciferase coding region were constructed. These constructs were introduced into aleurones of genotypes carrying either dominant or recessive alleles of the C1 and R genes, which are known to regulate anthocyanin production. Levels of luciferase activity in permissive backgrounds (C1, R) were 30- to 200-fold greater than those detected in tissue carrying one or both of the recessive alleles (c1, r) of these genes. These results show that genes delivered to intact tissues by microprojectiles are regulated in a manner similar to the endogenous genes. The transfer of genes directly to intact tissues provides a rapid means for analyzing the genetic and tissue-specific regulation of gene expression.

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system

1987 ◽  
Vol 7 (7) ◽  
pp. 2425-2434
Author(s):  
J M Heard ◽  
P Herbomel ◽  
M O Ott ◽  
A Mottura-Rollier ◽  
M Weiss ◽  
...  
Keyword(s):  
Transient Expression ◽  
Tissue Specificity ◽  
Transcriptional Start Site ◽  
Expression System ◽  
Hepatoma Cells ◽  
Sequence Motif ◽  
Specific Expression ◽  
Tissue Specific ◽  
Albumin Gene ◽  
Ccaat Box

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

scholarly journals Identification of aR2R3-MYBgene regulating anthocyanin biosynthesis and relationships between its variation and flower color difference in lotus (NelumboAdans.)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2369 ◽  
Author(s):  
Shan-Shan Sun ◽  
Paul F. Gugger ◽  
Qing-Feng Wang ◽  
Jin-Ming Chen
Keyword(s):  
Allelic Variation ◽  
Anthocyanin Biosynthesis ◽  
Population Analysis ◽  
Flower Color ◽  
Color Difference ◽  
Regulatory Genes ◽  
Anthocyanin Synthesis ◽  
Coding Region ◽  
Transcription Activator ◽  
Extant Species

The lotus (Nelumbonaceae:NelumboAdans.) is a highly desired ornamental plant, comprising only two extant species, the sacred lotus (N. nuciferaGaerten.) with red flowers and the American lotus (N. luteaWilld.) with yellow flowers. Flower color is the most obvious difference of two species. To better understand the mechanism of flower color differentiation, the content of anthocyanins and the expression levels of four key structural genes (e.g.,DFR,ANS,UFGTandGST) were analyzed in two species. Our results revealed that anthocyanins were detected in red flowers, not yellow flowers. Expression analysis showed that no transcripts ofGSTgene and low expression level of threeUFGTgenes were detected in yellow flowers. In addition, three regulatory genes (NnMYB5,NnbHLH1andNnTTG1) were isolated from red flowers and showed a high similarity to corresponding regulatory genes of other species. Sequence analysis ofMYB5,bHLH1andTTG1in two species revealed striking differences in coding region and promoter region ofMYB5gene. Population analysis identified threeMYB5variants inNelumbo: a functional allele existed in red flowers and two inactive forms existed in yellow flowers. This result revealed that there was an association between allelic variation inMYB5gene and flower color difference. Yeast two-hybrid experiments showed that NnMYB5 interacts with NnbHLH1, NlbHLH1 and NnTTG1, and NnTTG1 also interacts with NnbHLH1 and NlbHLH1. The over-expression ofNnMYB5led to anthocyanin accumulation in immature seeds and flower stalks and up-regulation of expression ofTT19inArabidopsis. Therefore, NnMYB5 is a transcription activator of anthocyanin synthesis. This study helps to elucidate the function ofNnMYB5and will contribute to clarify the mechanism of flower coloration and genetic engineering of flower color in lotus.

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes

1987 ◽  
Vol 7 (11) ◽  
pp. 3916-3922
Author(s):  
L J Mullins ◽  
G Veres ◽  
C T Caskey ◽  
V Chapman
Keyword(s):  
Methylation Status ◽  
Coding Region ◽  
Specific Expression ◽  
X Chromosomes ◽  
Partial Methylation ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Ornithine Carbamoyl Transferase ◽  
Inactive X Chromosome ◽  
Inactive Genes

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.

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