[cDNA cloning, subcellular localization and tissue expression of a new human Krüppel-like transcription factor: human basic Krüppel-like factor (hBKLF)].
The FLD4585 clone from the cDNA library of human fetal liver may encode a hematopoietic related transcription factor. Here we tried to clone its full-length cDNA from the 22 weeks-gestation human fetal liver and study its functional domains, genomic structure, chromosomal localization, subcellular site and expression pattern. To obtain the full-length cDNA of FLD4585 clone, 5' RACE technique was used. Bioinformatics was used to analyze its genomic structure, chromosomal localization and potential functional domains. Its subcellular localization was shown by GFP fusion technique. The expression pattern was studied by Northern blot, RT-PCR and Western blot. The results show the full-length cDNA encoded by FLD4585 clone is 1810 bp long and encodes a 345 amino acids protein with high homology to mouse BKLF (basic Krüppel-like factor). Its characteristic C-terminal three contiguous C2H2 zinc fingers place it within the family of Krüppel-like factors. Bioinformatics studies show hBKLF gene spans over 33 kb on chromosome 4p15.2-4p16.1 and contains 6 exons and 5 introns. GFP-hBKLF fusion technique showed hBKLF was present in the nuclei of COS-7 cells in a punctate pattern, whereas it was absent in the nucleoli. By Northern blot, hBKLF has two transcripts, one between 4.4 kb-7.5 kb and the other between 1.35 kb-2.4 kb. The larger transcript exists widely in human tissues. However, the smaller transcript was more restricted in blood leukocytes, liver and bone marrow. RT-PCR showed erythrocytes and granulocytes could both express hBKLF and its level increased as they matured. The expression level in fetal liver decreased as it developed towards adult liver and its hematopoietic function gradually lost. Taken together, in this paper we have successfully cloned the full length cDNA of hBKLF. Expression study suggests it may have broad functions in vivo, especially the functions in hematopoietic tissues.
Wang
Mang-Ju
MJ
Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Qu
Xiang-Hu
XH
Wang
Li-Sheng
LS
Zhai
Yun
Y
Wu
Shu-Lan
SL
He
Fu-Chu
FC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002874:Chromosome Mapping; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005260:Female; D020869:Gene Expression Profiling; D005796:Genes / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007438:Introns; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C501168:KLF3 protein, human; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins