Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes.

By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprI and sprII (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate.     

Regulation of the gene encoding the p24 actin-binding protein in Dictyostelium discoideum.

We have isolated cDNA clones on the basis of sequence similarity to the gene encoding the cyclic cAMP-binding protein CABP1 of Dictyostelium discoideum. The predicted amino acid sequence of the cloned cDNAs shows that the homology to CABP1 is restricted to a region rich in proline, glycine, glutamine, and tyrosine. Sequence comparison indicates that the cloned cDNAs encode the actin-binding protein p24. We have examined by RNA blot hybridization the expression of the gene encoding p24. For cells developed in suspension, the levels of p24 mRNA increase rapidly during early development, reaching a peak at 3-4 h. Addition of high concentrations of exogenous cAMP during the first 4 h of development produced little or no effect on the accumulation of p24 mRNA. Treatment with cAMP during subsequent stages of development reduced the levels of p24 mRNA. We attempted to determine if the synthesis of new proteins during early development is a requirement for the reduction in p24 mRNA levels by treating the cells with protein synthesis inhibitor. Unexpectedly, the addition of the inhibitor cycloheximide resulted in an increase in the level of p24 mRNA. The roles of cycloheximide and cAMP on the expression of the p24 gene are discussed.     

Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase

Antileukoproteinase (ALP) is a low mol wt mucosal secretory protein which, in human tissues, inhibits the activities of the neutral serine lysosomal proteinases elastase and cathepsin-G. In this study a number of recombinant cDNA clones corresponding to porcine ALP (pALP) were isolated from a cDNA library prepared from porcine endometrial poly(A)+ RNAs. The combined nucleotide sequences of the cDNA clones, representing the entire pALP mRNA sequence, are approximately 600 nucleotides long and encode a protein of 114 amino acids. The deduced amino acid sequence of pALP is 68% similar in primary structure to that of human ALP, is cysteine and proline rich, and exhibits a two-domain structure which, in the human protein, is involved in binding trypsin/cathepsin-G and elastase, respectively. However, pALP appears to lack the internal signal sequence of the corresponding human protein. Northern blot analysis of uterine RNAs using pALP cDNAs as probe demonstrated a single mRNA species approximately 0.8 kilobase in length. Uterine expression of pALP mRNA was highest in mid- and late pregnancy and very low or undetectable in early pregnancy. Estrogen and progesterone increased the levels of uterine pALP mRNA in prepubertal gilts, but not to the levels obtained at mid- and late gestation. pALP mRNA was also abundant in adult pig lung, where its expression was constitutive. Lower levels of pALP were found in fetal and neonatal lung and small intestine and in maternal cervix, spleen, and small intestine. Our study on the molecular cloning and analysis of pALP mRNA represents the first report on the porcine proteinase inhibitor and extends the identification of pregnancy-associated uterine proteins, which may play important functions in embryo or fetal development. The control of expression of pALP mRNA, which is distinct from those of other porcine uterine proteins studied to date, should provide additional insights into the mechanisms of regulation of uterine secretory activity.     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Primary structure of a human small nuclear ribonucleoprotein polypeptide as deduced by cDNA analysis

Anti-Sm is an antibody specificity often associated with the autoimmune disease systemic lupus erythematosus. The polypeptides Sm-B'/B (estimated molecular mass 27 and 26 kDa, respectively) are primary targets of Sm antibodies. Sm-B'/B are part of the core polypeptides of small ribonucleoprotein particles (snRNP) involved in pre-mRNA splicing. Sm-B'/B share the same amino-terminal sequence as we determined by microsequence analyses of the purified polypeptides. Oligonucleotide probes based on that sequence were used to isolate seven clones from a human lymphoblastoid cDNA library in lambda gt10. The clones contained a single coding region for a protein of approximately 25 kDa. The predicted amino-terminal sequence was identical to that of the isolated Sm-B'/B polypeptides. In vitro translation experiments produced a protein immunoreactive with human polyclonal anti-Sm antibodies. The isolation of only one unique cDNA sequence suggests that Sm-B'/B may be post-translational variants encoded by a single message. The specific structural features which distinguish Sm-B' from Sm-B have yet to be determined. Northern blot analysis confirmed the diverse tissue and species distribution expected for these immunologically conserved polypeptides. The Sm-B'/B primary sequence is rich in proline (20%) and glycine (15%) residues. The prolines are concentrated in the carboxyl-terminal half of the protein and display a repetitive unit that is shared with other snRNP and nucleic acid binding proteins. Analysis of these arrays suggests an eight residue proline-rich consensus sequence with potential as either an RNA binding domain, or as a site of protein/protein interaction.     

Characterization of a gene family encoding abscisic acid- and environmental stress-inducible proteins of alfalfa.

The phytohormone abscisic acid (ABA) has been proposed as a common mediator controlling adaptive plant responses to a variety of environmental stresses, including water deficit, salinity, wounding, and low temperature. We have recently isolated three cDNAs, pUM90-1, pUM90-2, and pUM91-4, from a cDNA library of ABA-induced mRNAs of alfalfa. These cDNA clones exhibit a very high degree of sequence homology with one another and sequence similarities with certain regions of several stress- and ABA-inducible genes. The polypeptides encoded by these cDNAs are very rich in glycine (35-40%), histidine (7-15%), asparagine (8-14%), and tyrosine (5-10%) and have no tryptophan and proline. All of the encoded polypeptides contain characteristic tandem repeats comprising glycine residues intercepted with histidine and/or tyrosine. The RNAs corresponding to a representative cDNA, pUM90-1, were induced after treatment of seedlings with low temperature, drought, salt, and wounding stress, but not by heat; the induction was maximal under low temperature treatment. ABA and ABA analog rapidly induced the expression of these genes, whereas gibberellic acid treatment exhibited no induction whatsoever. These genes appear to be specifically induced in the shoot tissues. Analysis of ABA induction of genes corresponding to pUM90-1 in alfalfa seedlings of different age groups demonstrated that these genes were inducible in seedlings/plants of all age groups examined. Taken together these results suggest that these cDNA clones encode a group of proteins that are inducible by ABA and multiple environmental stresses and correspond to a new family of genes of plants, designated as ABA- and environmental stress-inducible genes.     

Structure and expression of the cAMP cell-surface receptor

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.     

Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou

Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.     

Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation

During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.