6ene, 86 (1990) 241-249 Elsevier
241
GENE 03387
Organization and sequence of the human g e m encoding cytokeratin 8 (Recombinant DNA; introns; gene expression; cytoskeleton; intermediate filaments; keratins)
Stefan Krauss and Werner W. Franke Institute of Cell and Tumor Biology. German Cancer Research Center. D-6900 HeMelberg (F.R.G.)
Received by H.G. Zachan: 8 August 1989 Accepted: 18 September 1989
SUMMARY
Among the various intermediate filament (IF) proteins, cytokeratin 8 (CKg) is especially remarkable as it is produced early in embryogenesis, is the only type-II CK occurring in many simple epithelial cells, and can also be synthesized in certain non-epithelial cells. Using a eDNA probe specific for human CK8 we have isolated an approx. 14-kbgenomic clone (in phage 3.EMBL3) which contains the gene encoding human cKg. The gene comprising a total of 7766 nucleotides (nt) from the transcription start point, determined by primer extension analysis, to the polyadenylation site, determined from eDNA sequencing, and a 1030-nt 5' upstream region have been sequenced. The sequence of 485 amino acids (aa) deduced from the exon sequences (M, 53 532) shows strong homology with the corresponding gene products of bovine, murine and amphibian (Xenopuslaevis)origins. Surprisingly, the ck8 gene contains only seven introns instead of eight as found in all other genes of the same (type-II) CK subfamily; while all seven introns occur in identical positions as in the other type-ll CK-encoding genes, intron V of these genes is missing in the ck8 gene. lntron I of ck8 is remarkably long (2534 nt) and contains a cluster ofpotentiai regulatory sequences, including three Spl sites, and an extended Alu-element. In Southern-blot analyses, we found only one intron-containing gene encoding CK8 in the human genome, and by heterologous transfection experiments we showed that this 8ene is correctly transcribed in non-human cells expressing the orthologuus gene. The isolation and characterization of the ck8 gene provide the necessary basis for studying its chromosomal environment and the principles of regulation of its expression.
INTRODUCTION
In most vertebrate cells a large proportion of the cytoskeleton is represented by filaments formed by members of the muitigene family of IF proteins (Weber and Geisler, Correspondence to: Dr. W.W. Franke, Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg (F.R.G.) Tel, 06221/484212; Fax 06221/401271.
Abbreviations: aa, amino acid(s); bp, base pair(s); c/cg, gene (DNA) encoding cKg; CK, cytokeratin; GFP, 81iai filament protein; IF, intermediate filament(s); IgG, immunoglobulin G; kb, kilobase(s) or 1000 bp; NF, neurofilament; nt, nucleotide(s); oli8o, oligodeoxyribonucleotide(s); pas, polyadenylation site; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/0.015M Na3.citrate pH 7.6; SV40, simian virus 40; tsp, transcription start point; u, unit(s). 0378-1119/90/$03.50© 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
1984; Steinert and Roop, 1988). In human tissues thi~, family comprises more than 40 genes, including at least 29 genes encoding CK polypeptides (19 epithelial ones and another ten that are characteristic for trichocytes, i.e., hairand nail-forming cells; Franke et al., 1989). The various types of IF proteins share only little sequence homology ( ~
241
GENE 03387
Organization and sequence of the human g e m encoding cytokeratin 8 (Recombinant DNA; introns; gene expression; cytoskeleton; intermediate filaments; keratins)
Stefan Krauss and Werner W. Franke Institute of Cell and Tumor Biology. German Cancer Research Center. D-6900 HeMelberg (F.R.G.)
Received by H.G. Zachan: 8 August 1989 Accepted: 18 September 1989
SUMMARY
Among the various intermediate filament (IF) proteins, cytokeratin 8 (CKg) is especially remarkable as it is produced early in embryogenesis, is the only type-II CK occurring in many simple epithelial cells, and can also be synthesized in certain non-epithelial cells. Using a eDNA probe specific for human CK8 we have isolated an approx. 14-kbgenomic clone (in phage 3.EMBL3) which contains the gene encoding human cKg. The gene comprising a total of 7766 nucleotides (nt) from the transcription start point, determined by primer extension analysis, to the polyadenylation site, determined from eDNA sequencing, and a 1030-nt 5' upstream region have been sequenced. The sequence of 485 amino acids (aa) deduced from the exon sequences (M, 53 532) shows strong homology with the corresponding gene products of bovine, murine and amphibian (Xenopuslaevis)origins. Surprisingly, the ck8 gene contains only seven introns instead of eight as found in all other genes of the same (type-II) CK subfamily; while all seven introns occur in identical positions as in the other type-ll CK-encoding genes, intron V of these genes is missing in the ck8 gene. lntron I of ck8 is remarkably long (2534 nt) and contains a cluster ofpotentiai regulatory sequences, including three Spl sites, and an extended Alu-element. In Southern-blot analyses, we found only one intron-containing gene encoding CK8 in the human genome, and by heterologous transfection experiments we showed that this 8ene is correctly transcribed in non-human cells expressing the orthologuus gene. The isolation and characterization of the ck8 gene provide the necessary basis for studying its chromosomal environment and the principles of regulation of its expression.
INTRODUCTION
In most vertebrate cells a large proportion of the cytoskeleton is represented by filaments formed by members of the muitigene family of IF proteins (Weber and Geisler, Correspondence to: Dr. W.W. Franke, Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg (F.R.G.) Tel, 06221/484212; Fax 06221/401271.
Abbreviations: aa, amino acid(s); bp, base pair(s); c/cg, gene (DNA) encoding cKg; CK, cytokeratin; GFP, 81iai filament protein; IF, intermediate filament(s); IgG, immunoglobulin G; kb, kilobase(s) or 1000 bp; NF, neurofilament; nt, nucleotide(s); oli8o, oligodeoxyribonucleotide(s); pas, polyadenylation site; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/0.015M Na3.citrate pH 7.6; SV40, simian virus 40; tsp, transcription start point; u, unit(s). 0378-1119/90/$03.50© 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
1984; Steinert and Roop, 1988). In human tissues thi~, family comprises more than 40 genes, including at least 29 genes encoding CK polypeptides (19 epithelial ones and another ten that are characteristic for trichocytes, i.e., hairand nail-forming cells; Franke et al., 1989). The various types of IF proteins share only little sequence homology ( ~
