Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.     

Identification of rat cDNA encoding hck tyrosine kinase from megakaryocytes.

A rat homologue of hck tyrosine kinase cDNA from a rat megakaryocyte library contains 1911 nucleotides with an open reading frame encoding 503 amino acids. The rat hck had distinct amino acid residues from the mouse homologue exhibiting 97.6% identity. The sequence contains the SH2 and SH3 regions that interact with cytoplasmic signaling proteins, the kinase domain including the nucleotide binding site and the autophosphorylation site, and the C-terminal Tyr-499 known as a negative regulator.     

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.     

Cloning and analysis of coding cDNA sequence of human leukocyte Csk tyrosine kinase under normal conditions and in choroidal melanoma

The main function of Csk tyrosine kinases is phosphorylation of the C-terminal part of Src tyrosine kinases as a mechanism of their downregulation. A decrease in the expression of csk gene results in the enhancement of Src tyrosine kinase activity. In this study, cDNA containing the full coding sequence of the human leukocyte Csk tyrosine kinase gene has been cloned. The protein encoded by a 1624-bp cDNA fragment has 99% homology to human Csk tyrosine kinase. A comparative sequence analysis of full-length cDNAs for Csk tyrosine kinase of normal lymphocytes and lymphocytes of patients with choroidal melanoma revealed a nucleotide substitution in exon 10 of the gene, which appears to be of diagnostic significance. It has been shown that the risk of choroidal melanoma correlated with the frequency of this allele.     

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.     

Isolation and expression of a full-length cDNA encoding the human GM2 activator protein

We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Isolation and sequence analysis of a full-length cDNA for human M creatine kinase

A full length cDNA for human M creatine kinase has been isolated and sequenced. The cDNA contains 77 bp of 5' untranslated, 338 bp of 3' untranslated sequence and the entire coding region (1146 bp) for human M creatine kinase. The M creatine kinases from different species share considerable sequence homology within the coding region (77-91%) and in amino acid sequence (82-97%). Little or no sequence homology is observed in the 3' untranslated sequence of the mammalian M creatine kinases, although canine and human creatine kinase share overall 80% sequence homology in 5' untranslated sequence. A unique 8 bp sequence was identified in the 5' untranslated regions of mammalian M creatine kinase but is not present in B creatine kinase cDNA. The degree of sequence conservation observed implies an evolutionary constraint on M creatine kinase structure beyond that which would be expected for the maintenance of enzymatic function.     

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.     

Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppol) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression ofsugppol were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated thatsugppol was significantly different to PPO genes in C3 dicotyledonous plants.     

Selection of cDNAs encoding putative type II membrane proteins on the cell surface from a human full-length cDNA bank

We have developed a simple method to test whether a hydrophobic segment near the N-terminus of a protein functions as a type II signal anchor (SA) in which the N-terminus faces the cytoplasm. A cDNA fragment containing the putative SA sequence of a target clone was fused in-frame to the 5' end of a cDNA fragment encoding the protease domain of urokinase-type plasminogen activator (u-PA). The resulting fused gene was expressed in COS7 cells. Fibrinolytic activity on the cell surface was measured by placing a fibrin sheet in contact with the transfected COS7 cells after removing the medium. When the cDNA fragment encoded a SA, the fibrin sheet was lysed by the u-PA expressed on the cell surface. The fibrinolytic activity was not detected in the culture medium, suggesting that the u-PA remains on the cell surface anchored via the SA in the membrane without being cleaved by signal peptidase. This fibrin sheet method was successfully applied to select five novel cDNA clones encoding putative type II membrane proteins from a human full-length cDNA bank.     

Prediction of human cDNA from its homologous mouse full-length cDNA and human shotgun database

We propose a prediction method for human full-length cDNA by comparing sequence data between human genome shotgun sequence and mouse full-length cDNA. The human genome which is homologous to the mouse full-length cDNA is selected by a homology search program, and the predicted exons are connected at the exon-intron junction which gives the best homology score to the mouse full-length cDNA. The accuracy of the predicted human full-length coding region is 83.3%, and the false positive rate is 16.7%. Five human full-length proteins out of 20 proteins are correctly predicted.