The growth hormone-encoding gene isolated and characterized from Labeo rohita Hamilton is expressed in CHO cells under the control of constitutive promoters in 'autotransgene' constructs.
The growth hormone (GH) gene along with its regulatory sequences has been isolated from the blood and pituitary gland of Labeo rohita. This GH gene is approximately 2.8 kb long and consists of five exons and four introns of varying sizes with AG/TA in its exon-intron junctions. The promoter has a single cyclic AMP response unit (CRE) element, TATA, CAT and several Pit 1 binding sequences. The 1169-bp gene transcript starts 54 bp upstream of the ATG initiation codon and has two polyadenylation signals, ATTAAA, after the TAG stop codon. The mature mRNA has the poly (A) tail inserted 16 bp downstream of the second polyadenylation signal. Four chimeric 'autotransgenes' were constructed having either histone 3 or beta-actin promoter and cDNA or the total GH gene. The functionality of the individual components of the autotransgene was determined in the Chinese hamster ovary (CHO) cells by transfection experiments. Based on the results, the transcription of the GH gene is initiated at the transcription start signal of the respective promoters and terminates at the 3' regulatory sequence of the GH gene. Expression of GH in CHO cells shows that the fish promoters are active, the splicing signal is recognized, and the mRNA produced is stable and translated. The GH protein produced is effectively translocated and secreted into the medium. These results indicate the usefulness of CHO cells in determining the property of individual components of autotransgenes constructed from L. rohita and overall functional commonality between fish and mammal.
Rajesh
R
R
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India.
Majumdar
K C
KC
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003530:Cyprinidae / Q000235:genetics*; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D013006:Growth Hormone