The evolution and complexity of the genes encoding the cytoskeletal proteins of human epidermal cells

In order to explore the differential expression and evolutionary conservation of the genes encoding the cytoskeletal proteins for human epidermal cells, we have constructed a library of bacterial plasmids containing inserts of double-stranded cDNAs complementary to the mRNAs of cultured human epidermal cells. Cloned hybrid plasmids containing 45-95% of the sequences present in keratin, actin, and tubulin mRNAs were isolated and characterized. To identify the cDNAs encoding the four major keratins of human epidermal cells, the clones were initially screened for their ability to hybridize strongly with 32P-labeled cDNA prepared from unfractionated epidermal mRNA (about 30% keratin mRNA). Strongly hybridizing clones were further characterized by positive hybrid selection. Two distinct classes of clones were identified: One class hybridized specifically to the 56 and 58kd keratin mRNAs and one class hybridized specifically to the 46 and 50kd keratin mRNAs. Each class is encoded by a separate multigene family of about 10 genes. The two classes of sequences are conserved throughout vertebrate evolution, indicating the functional importance of each class in filament assembly. Clones containing human cDNA sequences encoding a cytoplasmic actin and alpha-tubulin were selected by hybridization screening using 32P-labeled cloned cDNAs for the mRNAs of beta-actin and alpha-tubulin of embryonic chick brain. The identity of these clones was established by positive hybrid selection and by DNA sequence analysis. Similar to the keratins, the actins and tubulins are also encoded by multigene families which are highly evolutionarily conserved. The availability of cloned cDNAs specific for each of three types of epithelial cytoskeletal proteins allows us to investigate the coordinate expression of their mRNAs during terminal differentiation in human epidermis.