Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.     

Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristics J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated resides characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.     

Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H(+)-ATP synthase

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H(+)-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits.     

Emotions, coping and the need for support in families of children with cancer: a model for psychosocial care

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.     

Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta

This paper presents the first purification of the mitochondrial branched-chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82-85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched-chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched-chain amino acid catabolism is specific to each species.     

Expression of a novel rat protein tyrosine phosphatase gene

The cDNA sequence and expression of a novel rat protein tyrosine phosphatase (PTP) gene is reported. The predicted amino acid sequence is similar to rat PRL-1, but is more closely related to human PTP4A, another member of the recently identified fourth group of PTPs. Therefore, multiple PTPs of this group are expressed in mammalian species. The novel rat PTP gene is expressed in the anterior pituitary gland in a sexually dimorphic pattern which is indicative of a specialized role in endocrine function.     

Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5

Outer mitochondrial membrane cytochrome b5 (OMb), which is an isoform of cytochrome b5 (cyt b5) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b5, in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.     

Molecular cloning of a bovine ornithine decarboxylase cDNA and its use in the detection of restriction fragment length polymorphisms in Holsteins

A cDNA coding for ornithine decarboxylase (ODC) was isolated from a bovine liver cDNA library. The clone (1758 base pairs) consisted of 5'- and 3'-untranslated regions of 185 and 187 nucleotides, respectively, and an open reading frame of 1383 nucleotides encoding an ODC protein (M(r) 51,342 daltons) of 461 amino acids. Comparison of the nucleotide and the predicted amino acid of the cDNA with other mammalian ODCs showed a very high degree of homology both at the DNA and protein levels. The bovine ODC mRNA was identified by northern blot to be a single species with a molecular size of 2.35 kilobase pairs. Primer extension analysis indicated that the 5'-untranslated region of the bovine ODC mRNA was 312 nucleotides long. Southern blot analysis of bovine genomic DNA revealed restriction fragment length polymorphisms when cleaved with restriction enzymes PstI, MspI, TaqI, and Bg/I.     

Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney

We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main. N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.     

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Polarographic detection of reoxygenation of lymph node metastases in the initial phase of primary radio- and radiochemotherapy in patients with advanced squamous epithelial carcinomas of the head and neck

Tyrosine phosphorylation is widely recognized as playing an important role in cell differentiation, proliferation and carcinogenesis. We used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that were expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen neonatal murine skin mRNA for clones encoding amino acid contiguities, the conservation of which is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, a novel clone encoding protein tyrosine kinase, PTK70, was identified. A full-length cDNA was isolated from a mouse thymus cDNA library. The nucleotide and deduced amino acid sequence showed that it featured src-homology (SH) 2 domain, SH3 domain and kinase domain like other src family protein tyrosine kinases, but lacked the N-terminal myristylation site and C-terminal tyrosine residue. Although the mRNA of PTK70 was detected in various tissues ubiquitously, the degree of its expression differed among tissues. Murine skin is one in which PTK70 was expressed strongly, with its expression being much stronger in the epidermis and in the cell line derived from murine keratinocytes than in those from melanoma or fibroblast cell lines. These evidences suggest that PTK70 may be involved in proliferation or differentiation of keratinocytes in the skin.     

Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.     

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.     

The expression of steroidogenic acute regulatory protein (StAR) in bovine adrenocortical cells

StAR protein may facilitate rapid transfer of cholesterol from the outer to the inner mitochondrial membrane, the site at which cholesterol is converted to pregnenolone by the cholesterol side chain cleavage complex. We have studied the effect of ACTH treatment on StAR mRNA and protein levels in bovine adrenocortical cells in primary culture. Cells were initially cultured for 3 days after isolation, and then treated with ACTH (10(-8) M) for various times up to 24 hours. Northern analysis of total BAC mRNA, using a [alpha32P]-labelled cDNA probe encoding a 5' region of bovine StAR mRNA, revealed two principal hybridising species of 1.6 and 3.0 kb. Western immunoblot analysis revealed a principal band at 30 kDa. Levels of both StAR mRNA and protein showed an increase at 1 hour, reached a maximum at around 6 hours and declined to basal levels at 24 hours. Cortisol secretion (measured by RIA) showed a similar change over the same period. From these results it appears that StAR mRNA and protein levels in BAC are acutely regulated in concert with ACTH-stimulated cortisol secretion.     

A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor

We describe here evidence of congenital enzyme mistargeting induced not by abnormalities in the signal sequence. We examined the molecular mechanism of hereditary ornithine aminotransferase (OAT) deficiency causing gyrate atrophy of the choroid and retina (GACR). Nucleotide sequencing of OAT cDNA generated from a GACR patient's mRNA revealed a single base change from C to G at position 268, resulting in an amino acid substitution of neutral Gln(CAA) with negatively charged Glu(GAA) at position 90 (Q90E). Immunohistochemical and transient expression analyses suggested expression of a defective labile OAT in the patient's tissues. However, high-level expression and immunocytochemical analyses elucidated that Q90E OAT (the patient's OAT) was localized within the limits of cytoplasmic free ribosomes in precursor form without any mitochondrial entry, indicating that the patient's precursor OAT was synthesized and rapidly degraded because of accumulation in the cytosol. It is interesting that, although the mutation site (Q90E) in this GACR patient's OAT was within the coding sequence of the mature protein, the precursor exhibited loss of mitochondrial targeting function. These findings suggest that not only the signal sequence but a critical part of the mature sequence plays an essential role in mitochondrial entry of the OAT precursor protein.     

Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain

We have identified a new calcium-dependent subfamily of mitochondrial carrier proteins with members in Saccharomyces cerevisiae, Caenorhabditis elegans, and various mammalian species. The members of this subfamily have a bipartite structure: a carboxyl-terminal half with the characteristic features of the mitochondrial solute carrier superfamily and an amino-terminal extension harboring various EF-hand domains. A member of this subfamily (that we have termed Aralar) was cloned from a human heart cDNA library. The corresponding cDNA comprises an open reading frame of 2037 base pairs encoding a polypeptide of 678 amino acids. The carboxyl-terminal half of Aralar (amino acids 321-678) has high similarity with the oxoglutarate, citrate, and adenine nucleotide carriers (28-29% identity), whereas the amino-terminal half (amino acids 1-320) contains three canonical EF-hands. Aralar amino-terminal half was shown to bind calcium by 45Ca2+ overlay and calcium-dependent mobility shift assays. The subcellular localization of the protein in COS cells transfected with Aralar was exclusively mitochondrial. Antibodies against Aralar amino-terminal fusion protein recognized a 70-kDa protein in brain mitochondrial fractions. Northern blot analysis showed that the protein was expressed in heart, brain, and skeletal muscle. The domain structure, mitochondrial localization, and presence in excitable tissues suggests a possible function of Aralar as calcium-dependent mitochondrial solute carrier.