Molecular cloning of a cDNA for the catalytic subunit of rabbit muscle phosphorylase phosphatase

A cDNA coding for the catalytic subunit of phosphorylase phosphatase (phosphatase C-I/phosphatase-1c) was cloned from a rabbit muscle cDNA library by screening with oligonucleotide probes. Ten clones were analyzed. The full cDNA sequence of 1395 base pairs contained an open reading frame of 990 base pairs flanked by 3' and 5' noncoding regions of 84 and 321 base pairs, respectively. The DNA sequence (and deduced amino acid sequence) of this cDNA is distinctly different from that of a clone of 1492 base pairs previously reported. Our cDNA is essentially identical to the 1492-base pair clone from residue 182 in the 3' direction, but it is completely different in the 5' direction. Consequently, the amino acid sequence deduced from our cDNA differs by 14 amino acids in the amino terminal from that previously reported and extends for an additional 19 amino acids. Probes to the divergent and common region of our cDNA clone hybridized to an mRNA of the same size by Northern blotting. Thus the cDNA we have isolated appears to code for an isoform of the catalytic subunit of phosphorylase phosphatase.     

The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells

A cDNA library was constructed in the phage lambda gt11 from human liver mRNA enriched for plasminogen mRNA by chromatography on Sepharose 4B. A full-length cDNA clone of human plasminogen was isolated. The 2.7 kb cDNA encoded the entire plasminogen molecule, a signal peptide sequence and two start codons with a 5'-untranslated region of about 80 base pairs. In the 3'-non coding region of 280 base pairs a consensus signal AATAAA was found at a distance of 46 base pairs upstream of the poly(A) tail. The plasminogen cDNA was subcloned in the eukaryotic expression vector p91023 (B), and human plasminogen was expressed in monkey kidney (COS m6) cells and in Escherichia coli. The recombinant molecule obtained from COS cells has physicochemical and biological properties similar to native human plasminogen I, indicating that it has folded in a manner similar to plasminogen synthesized by liver. By contrast, plasminogen expressed in E. coli could not be activated and showed biological properties which are very different from glycosylated forms of plasminogen. However, the non-glycosylated plasminogen was bound by lysine-Sepharose and reacted with a conformation dependent monoclonal antibody to kringles 1 to 3. These data suggest that the protein has properly folded kringle domains. Our studies suggest that the carbohydrate domains may play an important role in the function of the plasminogen molecule.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein S11

A cDNA clone specific for rat ribosomal protein S11 was isolated by hybrid-selected translation from the cDNA library made for 8-9 S poly(A) RNA from regenerating rat liver. Since this cDNA had not enough length, another clone was selected by colony hybridization using a fragment of isolated cDNA as a probe. The nucleotide sequence of the cDNA was determined. The sequence contains 2 base pairs from the 5' noncoding region, the entire coding region of 477 base pairs, and the 3' noncoding region of 55 base pairs besides the poly(A) tail. The primary structure of the protein S11 was deduced from the nucleotide sequence. It consists of 157 amino acids. Its molecular weight is 18,299. The calculated amino acid composition is consistent with the reported composition of S11 determined on the protein hydrolysate. The amino acid sequence showed a marked homology with that of S16 of Halobacterium cutirubrum and an appreciable homology with that of S17 of Escherichia coli.     

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.     

Molecular cloning of a bovine cathepsin.

A cDNA clone for a thiol endoproteinase has been isolated from a bovine heart cDNA library by using a mixture of 32 synthetic oligonucleotides as a hybridization probe. The inserted region is 672 base pairs in length. It contains a sequence encoding the C-terminal region of a protein that is homologous to rat liver cathepsins B and H and to plant thiol proteinases. In addition, it contains the sequence of 442 bases corresponding to the 3' untranslated region of the mRNA. The inserted region was used as a specific probe in RNA transfer analysis; the size of the mRNA encoding the thiol endoproteinase is estimated to be approx. 1.7 kilobases. Thus, the maximum size of the encoded protein is about 350-400 amino acids.     

Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic-acid-binding protein and chromosomal localization of the corresponding human gene

A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5' untranslated portion, the whole translated and the complete 3' untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.     

Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.     

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.     

Guinea-pig casein A cDNA. Nucleotide sequence analysis and comparison of the deduced protein sequence with that of bovine alpha s2 casein

The nucleotide sequence (1036 bases) of guinea-pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5' noncoding region, and the complete coding and 3' noncoding region. The remaining 5' noncoding sequence was obtained by primer extension. The deduced 223-amino-acid-coding sequence of guinea-pig pre-casein A exhibited 30% homology with bovine alpha s2 casein, the most striking similarities being in the locations of potential phosphorylation sites.     

Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene

The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

A marsupial phosphoglycerate kinase (PGK) processed pseudogene

A clone that cross-hybridized with a full-length human cDNA PGK probe was isolated from a hill kangaroo (Macropus robustus: Marsupialia) lambda EMBL4 EcoRI genomic library. The clone was sequenced and demonstrated to be a pseudogene, with two deletions (one of 3 bases, the other 24 bases long), one single base insertion, and a nonsense mutation with respect to the functional human X-linked gene. It is flanked by terminal repeats in the 5' and 3' noncoding regions, but it has no 3' poly(A) remnant. The 3' untranslated region has a 34-bp sequence, with 29 bp homologous to the human 3' untranslated region. The overall percentage homology with the mouse and human X-linked PGK indicates that this pseudogene is probably more closely related to eutherian X-linked PGK genes than to the autosomal form. The results also suggest that pseudogenes are of considerable antiquity (greater than 100 MYr) in the mammalian lineage.