Expression of two proopiomelanocortin genes in the pituitary gland of Xenopus laevis: complete structures of the two preprohormones.

A number of cDNA clones corresponding to Xenopus POMC mRNA was isolated from a cDNA library constructed from Xenopus pituitary polyadenylated RNA. Characterization of the cDNA inserts revealed two groups of structurally different proopiomelanocortin mRNAs, indicating that two proopiomelanocortin genes are expressed to virtually the same level in Xenopus pituitary glands. From the mRNA structures the complete amino acid sequences of the two Xenopus preproopiomelanocortins could be deduced. Comparison with proopiomelanocortin mRNA and protein sequences from other species shows regions of high homology (including the portion of the prohormone located N-terminally of gamma-melanophore-stimulating hormone) and regions of extremely low homology (including the signal sequence).     

Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species.

The Ku (p70/p80) autoantigen is a DNA-protein complex recognized by sera from certain patients with SLE and related diseases. Although human autoantibodies react with at least eight different epitopes of the human Ku complex, they had little reactivity with rodent Ku Ag on immunoblots. Small amounts of 70- and 80-kDa proteins were immunoprecipitated from murine cell extracts, however, suggesting that the Ku particle is not unique to human cells. This was confirmed by isolating cDNA clones encoding murine Ku Ag by plaque hybridization with a human p70 cDNA probe. The murine p70 cDNA clones had a deduced amino acid sequence 82.9% identical to that of human p70, and comparable amounts of murine and human p70 mRNA were detected in 3T3 and K562 cells, respectively. The poor reactivity of human autoantibodies with murine p70 was attributable to specific amino acid substitutions in an immunodominant conformational epitope located on amino acids 560-609 of human p70. Several amino acids critical for antigenicity of this region were defined by mutagenesis studies. Other conformational epitopes of Ku were also antigenically poorly conserved among species. Species-specific epitopes recognized by lupus autoantibodies are unusual but not unique to Ku. In general, poorly conserved autoepitopes have been conformational, rather than sequential, suggesting that the antigenicity of conformational epitopes may be particularly sensitive to evolutionary change.     

Primary structure of human, chicken, and Xenopus laevis p11, a cellular ligand of the Src-kinase substrate, annexin II.

The p11 protein is a member of the S-100 family of Ca(2+)-binding proteins and serves within the cell as a ligand of the tyrosine kinase substrate, annexin II. To obtain more structural information on this molecule, we have isolated and characterized p11 cDNA clones from several different species. A comparison of the deduced amino acid (aa) sequences reveals that mammalian and avian p11 are highly similar (at least 90% identical at the aa level), whereas p11 from Xenopus laevis shows a considerable degree of sequence variation (the aa sequence identity drops to approx. 60% when compared to mammalian or chicken p11). Interestingly, the C-terminal 18 aa, which are unique to p11 within the S-100 family, show a relatively high conservation among species. This high evolutionary conservation is in line with a structurally and/or functionally important role of this C terminus, e.g., in annexin II binding.     

Primary structure of bovine lactate dehydrogenase-A isozyme and its synthesis in Escherichia coli

Eleven cDNA clones encoding lactate dehydrogenase(LDH)-A isozyme were isolated from a bovine lymphocyte cDNA library, and the nucleotide sequences of three of the clones (pLDH5, pLDH9 and pLDH12) were determined. With the exception of variation in the 5' portion, two cDNA clones (pLDH9 and pLDH12) appeared to contain the full-length cDNA of 1786 bp, consisting of the protein-coding sequence (996 bp), the 5'- and the 3'-untranslated regions and the poly(dA) tail. The predicted amino acid (aa) sequence of bovine LDH-A (332 aa) showed 96.7% homology with that of pig LDH-A. The protein-coding cDNA region (1650 bp) was inserted into an Escherichia coli expression vector ptac11 and expressed. The protein synthesized in E. coli showed enzyme activity of LDH and was identified by cellogel electrophoresis as LDH-5 isozyme whose subunit M chain is the product of the LDH-A gene.     

Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato

Two pectin esterase cDNA clones representing different isozymes with ca. 95% homology were isolated from an early ripening tomato fruit cDNA library. Both clones were longer than previously published sequences, and the encoded proteins possessed extended (229–233 amino acid) putative N-terminal extensions. In addition, the mRNA species corresponding to the two clones showed differential levels of expression in fruit.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity

The Ku protein is a heterodimer composed of 70 kD (Ku70) and 80 kD (Ku80) subunits. Ku is the regulatory component of the DNA-dependent protein kinase (DNA-PK) that has a catalytic subunit of ~460 kD (DNA-PK_cs). In this study, the two polypeptides (Ku80/Ku70) of the human Ku were expressed in Xenopus oocytes in order to investigate their over-expression, sub-cellular localization, and functional interaction with the Xenopus DNA-PK_cs. In vitro-transcribed mRNAs for Ku70 and Ku80 were obtained from the respective plasmid constructs. The exogenously expressed proteins from the injected mRNAs were immunoprecipitated using a specific anti-T7 Tag antibody. The T7 Tag epitope is present in the vector at the amino-terminus and is in-frame with the Ku cDNA sequences. While injected Ku70 mRNA translated to a full-length Ku70 polypeptide that translocated to the nucleus, injected Ku80 mRNA resulted in the expression of a truncated product that was retained in the cytoplasm. Although Ku80 mRNA was stable for a period of 18 h in the oocytes post-microinjection, the protein was only stabilized when co-expressed with Ku70, suggesting that Ku80 is susceptible to proteolytic degradation when not dimerized with Ku70. Furthermore, the immunocomplex was capable of phosphorylating the DNA-PK-specific substrate thereby indicating that the holoenzyme could functionally reconstitute in vivo in the oocytes by heterologous subunits thus demonstrating evolutionary conservation of the enzyme subunit structure and function among diverse species.     

cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Primary structures of actinoporins from sea anemone Oulactis orientalis

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with specific primers to the N-terminal regions of the 0. orientalis actinoporin sequences and to the C-terminal region, which is known to be highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were established by sequencing the cDNA clones obtained by the fast amplification of 3'-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain lower hemolytic activities of Or-A and Or-G than that of actinoporins from the tropical species.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.     

Nucleotide sequence and genomic structure analyses of the p70 subunit of the human Ku autoantigen: evidence for a family of genes encoding Ku (p70)-related polypeptides

cDNA encoding the p70 polypeptide subunit of the human Ku autoantigen was isolated. In vitro expression analysis of the cDNA demonstrates that it encodes the entire open reading frame. Nucleotide sequence analysis and comparison to other previously described sequences indicate the existence of several single-nucleotide and amino acid polymorphisms. Southern blot analyses demonstrate the presence of multiple copies of homologous DNA sequences in the human genome. These data support the hypothesis that multiple genes encode a family of Ku(p70)-related polypeptides.     

Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80.

Ku, a heterodimer of approximately 70 and approximately 80 kDa subunits, is a nuclear protein that binds to double-stranded DNA ends and is a component of the DNA-dependent protein kinase (DNA-PK). Cell lines defective in Ku80 belong to group XRCC5 of ionizing radiation-sensitive mutants. Five new independent Chinese hamster cell mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B, that belong to this group were isolated. To shed light on the nature of the defect in Ku80, the molecular and biochemical characteristics of these mutants were examined. All mutants, except XR-V12B, express Ku80 mRNA, but no Ku80 protein could clearly be detected by immunoblot analysis in any of them. DNA sequence analysis of the Ku80 cDNA from these mutants showed a deletion of 252 bp in XR-V10B; a 6 bp deletion that results in a new amino acid residue at position 107 and the loss of two amino acid residues at positions 108 and 109 in XR-V11B; a missense mutation resulting in a substitution of Cys for Tyr at position 114 in XR-V13B; and two missense mutations in XR-V16B, resulting in a substitution of Met for Val at position 331 and Arg for Gly at position 354. All these mutations cause a similar, 5-7-fold, increase in X-ray sensitivity in comparison to wild-type cells, and a complete lack of DNA-end binding and DNA-PK activities. This indicates that all these mutations lead to loss of the Ku80 function due to instability of the defective protein.     

Expression of a novel cadherin (EP-cadherin) in unfertilized eggs and early Xenopus embryos

Two distinct cadherin cDNA clones of Xenopus laevis were isolated from a stage 17 embryo cDNA library. Analysis of the complete deduced amino acid sequences indicated that one of these molecules is closely homologous to chicken and mouse N-cadherin, while the other displays comparable homology to both E- and P-cadherins and was thus denoted EP-cadherin. This molecule has an apparent relative molecular mass of 125 x 10(3) (compared to approx. 138 x 10(3) or approx. 140 x 10(3) of E-cadherin and N-cadherins, respectively). Northern and Western blot analyses indicated that N-cadherin is first expressed at the neurula stage while EP-cadherin is the only cadherin detected in unfertilized eggs and cleavage stage embryos. Immunolabeling of Xenopus eggs with antibodies prepared against a fusion protein, containing a segment of EP-cadherin, indicated that the protein is highly enriched at the periphery of the animal hemisphere. EP-cadherin was also found in A6 epithelial cells derived from Xenopus kidneys, and was apparently localized in the intercellular adherens junctions.