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Function and regulation domains of a newly isolated putative β-actin promoter from pacific white shrimp.

Shi Y, Soderlund M, Xiang J, Lu Y - PLoS ONE (2015)

Bottom Line: The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif.Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1.Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5'-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (actinT1), has been isolated from the Pacific white shrimp (Litopenaeus vannamei) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5' regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and EGFP (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

No MeSH data available.


Related in: MedlinePlus

Putative-regulatory sequence of the β-actin gene homologues.Sequence alignment of the proximal promoter region is shown. Black and open boxes mark conserved sequences and known factor binding sites, respectively; CCAAT-box and CArG motif. GenBank accession number of compared fish β-actin genes are as follows: Oreochromis niloticus (AY116536.1); Common carp (C. carpio) (M24113.1).
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pone.0122262.g006: Putative-regulatory sequence of the β-actin gene homologues.Sequence alignment of the proximal promoter region is shown. Black and open boxes mark conserved sequences and known factor binding sites, respectively; CCAAT-box and CArG motif. GenBank accession number of compared fish β-actin genes are as follows: Oreochromis niloticus (AY116536.1); Common carp (C. carpio) (M24113.1).

Mentions: Our study has determined that the sequence located at -1642/-1325 of this putative shrimp promoter is an essential domain for the promoter activity. Despite the sequence of 5’-flanking region showing no close homologies to actins from other species, a stretch of 327 bp of proximal promoter was compared with that of Oreochromis niloticus and Common carp (C.carpio), and a putative transcription factor binding sites, CCAAT was identified at the location of -322 bp from transcriptional start site (TSS). In addition, a highly conserved CArG (CC (A/T) 6GG) motif was also identified at the location of -293 (Fig 6). The distances between the CCAAT and CArG boxes and the TSS are known to be variable, but they are usually within the first 220 bp from the Cap signal, upstream of the TATA box [24, 25]. In this study, a TATA box was predicted using the TFSEARCH system and was within the location of 140 bp. It has been proven that the CCAAT-box is an essential element for the promoter activity when joined to a heterologous gene [26] and CArG is an essential serum-response element positioned in the actin promoter region between the CCAAT and TATA box [27, 28].


Function and regulation domains of a newly isolated putative β-actin promoter from pacific white shrimp.

Shi Y, Soderlund M, Xiang J, Lu Y - PLoS ONE (2015)

Putative-regulatory sequence of the β-actin gene homologues.Sequence alignment of the proximal promoter region is shown. Black and open boxes mark conserved sequences and known factor binding sites, respectively; CCAAT-box and CArG motif. GenBank accession number of compared fish β-actin genes are as follows: Oreochromis niloticus (AY116536.1); Common carp (C. carpio) (M24113.1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383542&req=5

pone.0122262.g006: Putative-regulatory sequence of the β-actin gene homologues.Sequence alignment of the proximal promoter region is shown. Black and open boxes mark conserved sequences and known factor binding sites, respectively; CCAAT-box and CArG motif. GenBank accession number of compared fish β-actin genes are as follows: Oreochromis niloticus (AY116536.1); Common carp (C. carpio) (M24113.1).
Mentions: Our study has determined that the sequence located at -1642/-1325 of this putative shrimp promoter is an essential domain for the promoter activity. Despite the sequence of 5’-flanking region showing no close homologies to actins from other species, a stretch of 327 bp of proximal promoter was compared with that of Oreochromis niloticus and Common carp (C.carpio), and a putative transcription factor binding sites, CCAAT was identified at the location of -322 bp from transcriptional start site (TSS). In addition, a highly conserved CArG (CC (A/T) 6GG) motif was also identified at the location of -293 (Fig 6). The distances between the CCAAT and CArG boxes and the TSS are known to be variable, but they are usually within the first 220 bp from the Cap signal, upstream of the TATA box [24, 25]. In this study, a TATA box was predicted using the TFSEARCH system and was within the location of 140 bp. It has been proven that the CCAAT-box is an essential element for the promoter activity when joined to a heterologous gene [26] and CArG is an essential serum-response element positioned in the actin promoter region between the CCAAT and TATA box [27, 28].

Bottom Line: The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif.Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1.Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5'-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (actinT1), has been isolated from the Pacific white shrimp (Litopenaeus vannamei) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5' regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and EGFP (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

No MeSH data available.


Related in: MedlinePlus