Absorbed fraction to the cell nucleus for low energy electrons

The Drosophila dishevelled gene (dsh) encodes a secreted glycoprotein, which regulates cell proliferation, acting as a transducer molecule for developmental processes, including segmentation and neuroblast specification. We have isolated and characterized cDNA clones from two different human dsh-homologous genes, designated as DVL-1 and DVL-3. DVL-1 and DVL-3 putative protein products show 64% amino acid identity. The DVL-1 product is 50% identical to dsh and 92% to a murine dsh homologue (Dvl-1). Both human DVL genes are widely expressed in fetal and adult tissues, including brain, lung, kidney, skeletal muscle and heart. DVL-1 locus maps to chromosome 1p36 and DVL-3 to chromosome 3q27. DVL-1 locus on chromosome 1 corresponds to the murine syntenic region where Dvl-1 is located. DVL-1 and DVL-3 are members of a human dsh-like gene family, which is probably involved in human development. Although the precise role of these genes in embryogenesis is only conjectural at present, the structural and evolutionary characteristics suggest that mutations at their loci may be involved in neural and heart developmental defects.     

Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase

Here we report the cDNA sequence of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. The cDNA was isolated from a human placental cDNA library by screening with a 150bp probe generated by PCR using degenerate primers based on the sequences found in the sialyl motif. Comparative analysis of this cDNA with the rat liver alpha 2,3-sialyltransferase sequence indicates 91% nucleotide similarity between the two sequence in the predicted coding region. On the amino acid level, the degree of conservation is 97%. Surprisingly, Northern analysis indicated that the gene is expressed at low levels in human placenta but is abundantly expressed in skeletal muscle and fetal tissues.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

Molecular cloning of the cDNA encoding human skeletal muscle triadin and its localisation to chromosome 6q22-6q23

We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81,545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22-6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.     

Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis

Four closely related cyclic-nucleotide specific phosphodiesterase (PDE4) genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame (ORF) of 791 amino acids (aa) is encoded by PDE4C-791, which is similar to a recently described cDNA [Engels, P., Sullivan, M., Muller, T. and Lubbert, H. FEBS Lett. 358 (1995) 305-10], except that an alternative 5'-end sequence upstream of the first methionine extends the PDE4C-791 ORF by 79 aa. The PDE4C-426 variant contains 3 insertions that are located 5' to the catalytic domain and encode several in-frame stop codons. The predicted 426 aa protein initiates at a methionine 365 aa within PDE4C-791. A baculovirus clone starting at this methionine expressed an enzymatically active protein. Two additional splice variants, PDE4C-delta54 and PDE4C-delta109, were found in testis mRNA. PDE4C-delta54 contained a novel 5'-end region and a deletion of 162 nt; the predicted protein deletes 54 aa from the amino-terminal region. The PDE4C-delta54 protein produced in baculovirus-infected cells was enzymatically active and sensitive to PDE4-specific inhibitors. The PDE4C-delta109 protein is similar to PDE4C-delta54 but has an additional 55 aa deleted in the catalytic domain; it lacked enzymatic activity. Analysis of uncloned total mRNA from 4 tissue sources by polymerase chain reaction (PCR) confirmed the presence of mRNAs with the two deletions and three insertions that we observed in cDNA clones. The PDE4C-delta54 variant was found only in testis and the 5'-extended region of PDE4C-791 was seen only in lung and the melanoma cell line G361. Hence, tissue-specific expression of various PDE4C isoforms should be considered in understanding how these gene products modulate cellular responses to cAMP.     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5' untranslated region (5'-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3'-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn(2+)-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.     

Cloning and sequencing an ovine interleukin-4-encoding cDNA

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp in length and contains an open reading frame of 408 nucleotides (nt) coding for a 15.1-kDa IL4 precursor of 135 amino acids (aa). Cleavage of the putative signal peptide of 22 aa yields the mature form of 13.2 kDa. Analysis of the mature aa sequence shows two potential N-linked glycosylation sites and six Cys residues. Ovine and bovine IL4 are shorter than human, mouse and rat IL4, because of a 51-nt deletion in the coding region. Comparison of the predicted aa sequence shows that ovIL4 shares 92, 57, 37 and 42% identity with the bovine, human, mouse and rat IL4s, respectively.     

Cloning and characterisation of a novel P-glycoprotein homologue from barley

P-glycoproteins are members of a large superfamily of transport proteins (the 'traffic ATPases') that utilize ATP to translocate a wide range of substrates across biological membranes. Using a PCR-based approach, and degenerate oligonucleotides corresponding to conserved motifs, two 300-bp cDNA fragments (pBMDR1 and pBMDR2) with a significant sequence similarity to mammalian P-glycoproteins were amplified from barley (Hordeum vulgare) root poly A+ RNA and used as probes to screen a barley root cDNA library. A single full-length clone pHVMDR2 coding for a polypeptide of 1232 residues (c. 134 kDa) was isolated. Comparison of this barley sequence with Arabidopsis ATPGP1 and human MDR1 and MDR3 P-glycoprotein sequences showed that the barley cDNA has 44%, 37% and 38% amino acid (aa) identity, respectively, with these sequences, and conserved structural features. RNase protection analysis showed that HVMDR2 mRNA is expressed at low levels in both barley roots and leaves. Southern blot analyses indicated that there is a small multigene family related to P-glycoproteins in barley. Possible functions for these barley P-glycoproteins are discussed.     

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).     

Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.